Assays for trichomonal and other hydrolases

ABSTRACT

The release by trichomonads of a hydrolase that hydrolyzes a narrowly defined class of substrates at a low pH without interference from hydrolases that are unrelated to trichomoniasis is the basis for a selective diagnostic assay for trichomoniasis that measures hydrolysis of any of these substrates by vaginal fluid at a low pH. Selective assays for trichomoniasis are also obtained by removing particulate matter from a sample of vaginal fluid to extract a fraction devoid of particles greater than a selected size, and where desired, combining the extracted fraction with any of certain specified hydrolase inhibitors, then testing the fraction for enzymatic hydrolase activity. These qualities of trichomoniasis are the basis for a series of diagnostic tests and test devices that produce results that are detectable by visual and other means with a high degree of accuracy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of co-pending U.S. patentapplication Ser. No. 11/353,497 filed Feb. 13, 2006, which is acontinuation application of U.S. patent application Ser. No. 10/269,917,filed Oct. 10, 2002, and issued as U.S. Pat. No. 7,041,469 on May 9,2006, the contents of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention resides in the field of hydrolases (i.e., hydrolyticenzymes), and in particular, methods and devices for the detection ofhydrolase activity in a sample or specimen and the diagnosis of diseasebased on the detection of hydrolase activity. A specific area ofinterest of this invention is the detection of trichomoniasis in femalesubjects by assaying for the presence of enzymatically activetrichomonal hydrolases in a vaginal fluid specimen.

All literature cited in this specification, including patents, technicalarticles and books, are incorporated by reference in their entirety.

2. Description of the Prior Art

Trichomoniasis is a clinically important Sexually Transmitted Disease(STD) caused by the protozoan Trichomonas vaginalis. In 1955, the WorldHeath Organization estimated approximately 170 million new cases oftrichomoniasis would arise annually among adults worldwide, with higherprevalence and incidence rates in both developing and industrializedcountries than for any other sexually transmitted disease.Trichomoniasis is frequently undetected because most infected men andapproximately 50% of infected women are asymptomatic. Trichomoniasis inwomen may cause discomfort or a foul smelling vaginal discharge, and canbe associated with adverse clinical sequelae. Trichomoniasis canincrease the risk the human immunodeficiency virus (HIV) transmissionand infection (Laga et al., AIDS 7(1):95-102 (1993) and WHO PressRelease WHO/64 (1995)).

Trichomonads produce specific hydrolases called proteinases thathydrolyze proteins including, but not limited to, IgG, IgM, and IgAantibodies (Provenzano and Alderete, Infect. Immun. 63(9):3388-3395(1995)). These proteinases also damage secretory leukocyte hydrolaseinhibitor (SLPI), a protective factor normally present in vaginal fluidthat inhibits HIV viral entry into human monocytic cells (Draper et al.,J. Infect. Dis. 178(3):815-819 (1998)). Trichomonas vaginalis (T.vaginalis) is also thought to play a role in promoting cervical cancer(Yap et al., Genitourin. Med. 71(6):402-404 (1995)). Pregnant womeninfected with T. vaginalis at mid-gestation are more likely to have alow birth weight infant or to deliver preterm (Cotch et al., Sex.Transm. Dis. 24:353-360 (1997)).

The most common method for diagnosing trichomoniasis is wet mountmicroscopy, a microscopic examination of vaginal fluid specimens for T.vaginalis. This is a labor-intensive method which requires a microscopeand a skilled technician, and fails to detect approximately half of theinfected women (Baron et al., Laboratory Diagnosis of Female GenitalTract Infections, Cumitech 17A, American Society for Microbiology(1993)). Trichomoniasis can be diagnosed with high sensitivity byculturing vaginal fluid specimens, but this method requires culturemedia and long-term growth in a controlled-temperature incubator, inaddition to the use of a microscope and a skilled technician. Due to thelabor involved, the expense of culture media and supplies, the trainingrequired, and the delay of up to a week to detect trichomonal growth,few clinics or laboratories routinely use culture to diagnosetrichomoniasis.

A diagnostic test based on DNA amplification and detection has recentlybeen developed by Lawing et al., J. Clin. Microbiol. 38(10):3585-3588(2000). This test is sensitive but, like culture, not rapid enough toproduce the results during the patient's visit to the clinic. Due to thecost of the DNA-based test kits and the training and laboratoryequipment required to perform these tests, this methodology is notwidely used in clinics or in medical practice in general, particularlyin the parts of the world where the need for a trichomoniasis test isgreatest.

Thus, to date there is no rapid, accurate, cost-effective and simplemethod or test device for point-of-care diagnosis of trichomoniasis.Numerous studies have demonstrated that T. vaginalis produces a varietyof hydrolases; see for example Garber et al., Can. J. Microbiol.35:903-909 (1989). Two studies have demonstrated that vaginal fluidspecimens from women with trichomoniasis contain detectable levels ofhydrolases that disappear after infected women are treated and cured ofthe infection (Alderete et al., Genitourin. Med. 67(6):469 (1991), andGarber and Lemchuck-Favel, Parasitol. Res. 80(5):361-365 (1994)).Unfortunately, the laboratory-based hydrolase detection methods used inthe studies reported by these authors required equipment, skill andtraining and were labor-intensive and slow, so that the results wereobtained only after several hours. For example, the method used byAldrete et al. for detecting trichomonal hydrolases involved a four-stepprocess to produce gelatin-acrylamide zymograms: first, trichomonalhydrolases were electrophoretically concentrated into discrete bands ina sheet of polyacrylamide gel; second, the hydrolases were allowed todigest gelatin which had been immobilized within the polyacrylamide gel;third, the gel was stained with a general protein stain; and fourth, thegel was destained in a lengthy washing process which eventually revealedthe hydrolase-digested gelatin which appeared as clear bands on thedarkly stained background gel (the zymogram). Trichomonal hydrolaseswere detected in the Garber et al. study by polyacrylamide gelelectrophoresis followed by immunoblotting, a similarly complexprocedure that involved the use of rabbit antibodies to visualize aspecific hydrolase band. Both methods require a high level of expertiseand costly equipment and take many hours to complete, and are thereforeunsuitable for point-of-care testing.

The zymogram procedure described above can also be performed by usingsynthetic fluorogenic substrates to detect the trichomonal hydrolasesonce the hydrolases have been separated by gel electrophoresis. Thesesubstrates typically contain one or more amino acids linked to afluorogenic reporter group that becomes fluorescent only after it isenzymatically cleaved from the peptide group by a hydrolase.Unfortunately, this procedure still entails the unwieldy polyacrylamidegel electrophoresis step prior to testing for hydrolase activity withthe fluorogenic substrates. Moreover, hydrolysis of the substrate can beobserved only by examining the electrophoretic gels for bands thatfluoresce under ultraviolet light. Both the gelatin-digestion method andthe fluorogenic substrate method were utilized in a study ofintracellular and secreted T. vaginalis hydrolases reported by North etal., Mol. Biochem. Parasitol. 39:183 (1990). Several fluorogenicsubstrates were identified which could be used to detect trichomonalhydrolases. Unfortunately, the methods used in this study areimpractical for a point-of-care clinical diagnostic test, since gelelectrophoresis is slow and cumbersome and observation of fluorescencerequires instrumentation or a darkroom and ultraviolet illuminator. Thedifficulty is that vaginal fluid contains many different hydrolasessecreted by a variety of sources, including bacteria, which are presentat extremely high levels, white blood cells, vaginal epithelial cells,and others. Each method relies on electrophoretic separation to achieveselective detection of the trichomonal hydrolases. Withoutelectrophoretic separation, it could not be determined if thehydrolytically active bands were derived from trichomonads or from someother source of hydrolytic activity in a vaginal fluid specimen.

A trichomoniasis test is therefore needed that can be performed byattending clinicians quickly, simply, inexpensively and accurately whilethe patient is still present. It would be particularly beneficial to beable to perform the test with a disposable device that is inexpensiveand easy to use and one that rapidly produces accurate results.

SUMMARY OF THE INVENTION

A series of discoveries has now been made that permit a sample ofvaginal fluid to be tested for the presence of T. vaginalis in a fast,accurate, and efficient manner. These discoveries also lead to methodsand test devices for detecting the presence of enzymes in general,including various types of hydrolases, that are indicative of a varietyof physiological conditions. The specimens in which the detections areperformed may be any bodily fluids, vaginal fluid being but one example.

One discovery is that trichomonads release a particular hydrolase intovaginal fluid that actively hydrolyzes a narrowly defined class ofsubstrates at a low pH without interference from other hydrolases thatare unrelated to trichomoniasis and that are also present, or frequentlypresent, in vaginal fluid. A diagnosis of trichomoniasis can thus bemade by assaying vaginal fluid for hydrolytic activity against one ormore members of this class of substrates at low pH. Hydrolysis of thesubstrate is readily converted to a signal that is eithermachine-readable or visually detectable.

This discovery can be implemented in various diagnostic methods and testmaterials. One example is the use of an implement that is capable ofretaining liquid together with a solid support on whose surface aredeposited the assay components, in distinct regions if necessary,depending on the particular assay methodology. The implement can beapplied to the surface and the assay result can be read directly on orin the implement. The implement can for example be a swab, dropper,pipette, or other liquid transfer device, and the deposited assaycomponents can include a substrate and an indicator. Swabs areparticularly convenient implements since a swab can easily be wettedwith the sample, then rubbed on the operative surface of the solidsupport, causing the assay components deposited on the surface to adhereto the swab. This allows the user to perform the assay determination bysimply noting whether a detectable change, preferably a color change,has occurred on the swab.

A further discovery is that trichomonal hydrolase activity, withoutbeing restricted to low pH, can be separated from other sources ofhydrolase activity in a specimen of bodily fluid by size exclusion.Thus, by extracting from a specimen of bodily fluid, particularlyvaginal fluid, a fraction that is devoid of particulate matter above acertain size threshold, one can detect the presence or absence oftrichomoniasis in the specimen by determining whether hydrolyticcleavage of the substrate has occurred. Selectivity toward trichomonalhydrolase activity can be improved further by combining size exclusionwith the use of a narrowly defined class of hydrolase inhibitors. Thus,in preferred embodiments of this discovery, a sample of vaginal fluid isprocessed to extract a fraction that is devoid of particulate matterabove a certain size threshold and, in the presence of one or more ofthese inhibitors, the extracted fraction is brought into contact with anappropriate substrate to detect hydrolase activity. Restricting thesubstrate to a narrowly defined class provides even greater selectivityfor trichomonal hydrolase activity.

Related to the discovery addressed in the preceding paragraph is thediscovery of a test device that includes a migration path through whicha sample of bodily fluid that is applied to the device travels bycapillary force. The device contains porous material along the migrationpath to filter out particulate matter above a selected size threshold.The device is useful for the detection of any soluble enzyme that is notadhered to cells or other particulate matter in the bodily fluid, andbodily fluids on which the device can be used include vaginal fluid,urine, blood, saliva, and various others. The device also contains asubstrate that is acted upon by the enzyme of interest and an indicatorthat produces a signal as a result of action of the enzyme upon of thesubstrate. In assays for trichomoniasis, for example, the substrate willbe one that is hydrolyzed by trichomonal hydrolase activity. Analternative to the substrate-indicator combination is a substrate thatincludes a chromogen that undergoes a color change as a direct result ofthe action of the enzyme of interest. For enzymes that cause cleavage ofthe substrate, the chromogen may undergo a color change upon release ofthe chromogen from the remainder of the substrate. Hydrolases, includingtrichomonal hydrolases, are examples of such enzymes. Regardless ofwhether a substrate-indicator combination is used or a substrate is usedthat includes a chromogen whose color changes upon action of the enzyme,the substrate, the indicator if one is present, or both substrate andindicator are positioned far enough along the migration path that thevisual signal is attributable only to hydrolytic activity in thefiltered liquid. In preferred such devices, one of the class ofinhibitors referred to above is also included to further improve theselectivity of the assay.

Among the advantages that are offered by these discoveries, methods,compositions and devices are accuracy and selectivity in the diagnosisof T. vaginalis, tests that can be performed quickly with minimaltraining or instruction, and the ability to perform the tests in testdevices that are self-contained with all reagents included and requiringonly the application of the liquid test specimen. These and otherobjects, features, aspects, and advantages of the invention, as well asvarious embodiments of the principles forming the basis for the variousdiscoveries, will be apparent from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a vertical cross section of a test device in accordance withthis invention, in which the test result is read on the test deviceitself.

FIG. 2 is a vertical cross section of a second test device in accordancewith this invention, representing a variation of the test device of FIG.1.

FIGS. 3 a, 3 b, 3 c, and 3 d are vertical cross sections of a third testdevice in accordance with this invention, representing a furthervariation on the test devices of FIGS. 1 and 2.

FIG. 4 is a perspective view of a fourth test device in accordance withthis invention, in which the test result is observed on a swab that hasbeen wetted with a specimen of vaginal fluid and then applied to thedevice.

DETAILED DESCRIPTION OF THE INVENTION

In embodiments of the invention in which a sample of vaginal fluid isassayed for hydrolytic activity at low pH using a selective class ofhydrolase substrates, the low pH is preferably within the range of 2 to3.5, and most preferably within the range of 2.3 to 2.4. To perform theassay at a pH within these ranges, a sample of vaginal fluid musttypically be acidified to place its pH within these ranges, since thesample as first taken may have a pH anywhere within the range ofapproximately 3.8 to approximately 6.5. Acidification can beaccomplished at various different stages, such as acidifying the samplebefore contact of the sample with the substrate, acidifying thesubstrate before contact of the sample with the substrate, or acidifyingthe sample, substrate, or both while contacting the sample with thesubstrate. One method of acidification is dilution of the sample with anaqueous solution of an acidic buffer to bring the sample to the desiredpH, followed by contacting the acidified sample with the substrate. Theacidic buffer can be either in liquid form or in solid form, andexamples of acidic buffers are malic acid, glycine, lysine, andthreonine. Of these, malic acid and threonine are preferred. Aparticularly preferred buffer is 200 mM threonine adjusted to pH 2.3 or2.4.

In various embodiments of the invention, the substrate is a conjugateconsisting of a residue covalently bonded to a reporter group, thecovalent bond being cleavable by hydrolytic enzyme activity. The term“reporter group” is used herein to denote any group that creates,causes, or leads to the generation of a detectable signal upon releasefrom the residue. For the embodiments described in the precedingparagraph, the preferred conjugate includes as the substrate residue apeptide whose C-terminus is either arginine or lysine while the reportergroup is a species that produces a detectable change upon hydrolyticcleavage from the peptide, the hydrolytically cleavable covalent bondbeing at the C-terminus of the peptide. Examples of linkages thatcontain hydrolytically cleavable bonds are amide linkages and esterlinkages. The reporter group can thus be bonded to the substrate residuethrough either of these two linkages. The detectable change occurseither in the reporter group itself or in a separate indicator withwhich the reporter group comes into contact during the course of theassay. The peptide is preferably 1 to 6 amino acids in length, and mostpreferably 2 to 3 amino acids in length, and the preferred amino acid atthe C-terminus is arginine. To assure that the hydrolysis occurs at theC-terminus, the N-terminus of the peptide is preferably blocked with anN-blocking group. Examples of N-blocking groups are carbobenzoxy,benzoyl, t-butoxycarbonyl, and D-amino acids. Other examples will beapparent to those skilled in the art.

The detectable change can be either visually readable by a clinicianperforming the test or self-readable by one who is performing the teston oneself, or machine-readable by instrumentation that detects thechange and optionally performs additional functions, such as for examplecomparing the result to control values or to a calibrated scale,quantifying the result, performing two or more different tests on asingle specimen, or performing the same test simultaneously on amultitude of samples and developing either statistical data from theresults or simply recording them in a systemized manner.

The conversion that is caused by the cleavage of the reporter group fromthe substrate residue and that leads to the detectable change can beeither a chemical transformation or a spatial relocation. In embodimentswhere the detectable change results from chemical transformation, theinability of the reporter group to produce a detectable signal whencoupled to the substrate residue can for example be the result of achemical neutralizing effect of the residue on the reporter group, whicheffect is eliminated when the linkage is cleaved. In embodiments wherethe detectable change results from spatial relocation, one example isthe use of a substrate whose residue is permanently coupled to an inertsurface on the test device and an indicator that is also permanentlycoupled to an inert surface on the test device but at a locationspatially separated from the substrate residue. When a liquid sample isadded that wets both the substrate and the indicator and contains theenzyme of interest, the reporter group is cleaved from the substrateresidue and migrates through the liquid sample to the indicator toproduce the detectable change. Other examples will be readily apparentto those skilled in the art. In all of these examples, the cleavage ofthe linkage between the substrate residue and the reporter group canoccur by the direct action of the trichomonal hydrolase on the linkageor by the action of the trichomonal hydrolase in combination with otherhydrolases.

Reporter groups that produce a visually detectable color change arepreferred. One type of reporter group that produces a visuallydetectable color change is a compound that reacts with an indicator tocause a color change in the indicator. To assure that the color changeresults from hydrolytic cleavage of the reporter group from theremainder of the substrate (i.e., the substrate residue), the substrateand indicator can be immobilized at spatially separated locations in atest device in a manner rendering them insoluble in the sample, with thereporter group migrating or otherwise flowing toward the indicator onlyafter the reporter group has been cleaved from the substrate residue.Another type of reporter group that produces a color change is one thatitself changes color upon release from the substrate residue, i.e., uponcleavage of the covalent bond joining the reporter group to thesubstrate residue. After hydrolytic release from the substrate residue,the reporter group can be restrained or immobilized by chemical ormechanical means to confine the color signal in a defined region in thedevice.

Indicators that will display a color change when reacted with anappropriate reporter group include, but are not limited to,para-dimethylamino-cinnamaldehyde (pDMAC), diazonium salts, andtetrazonium salts. Examples of specific dyes within these classes areFast Garnet GBC, Fast Dark Blue G, Fast Red B, Fast Red RL, Fast CorinthV, Fast Bordeaux GB, Fast Violet B, and Fast Black K. Each of theseindicators is colorless or lightly colored in its unreacted state, andforms a highly colored derivative when reacted with reporter groups suchas phenols, naphthols, aromatic amines or structural analogs of suchreporter groups. Further examples of diazonium and tetrazonium salts anddescriptions of their use are found in Conn, H. J., Biological Stains,R. D. Lillie, M. D., ed., Baltimore: The Williams & Wilkins Co., NinthEdition (1977), pp. 200-224. Diazonium dyes are particularly usefulexamples.

As noted above, examples of linkages between the reporter group and thesubstrate residue that are cleavable by hydrolases are amide linkagesand ester linkages. The reporter groups can therefore be amines orhydroxyl compounds analogous to the amines. Examples of amines usefulfor this purpose are 4-methoxy-2-naphthylamine, β-naphthylamine, and7-amino-4-methylcoumarin. Examples of hydroxyl compounds useful for thispurpose are α-naphthol, β-naphthol, 3-hydroxy-2-naphthoic acid,6-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthalenesulfonic acid,1-naphthol-3,6-disulfonic acid, 6-bromo-2-naphthol, 6-hydroxy-2-naphthyldisulfide, and 4-hydroxy-1-naphthalenesulfonic acid.

Amino acids can serve as reporter groups that can produce detectablechanges in a wide variety of indicators, either directly or indirectly.Examples of indirectly generated color changes are chromogenic systemsin which the amino acid reporter group reacts with oxygen in thepresence of an amino acid oxidase to produce hydrogen peroxide and anoxidized amino acid, and the hydrogen peroxide then reacts with areduced chromogen in the presence of a redox catalyst to produce color.Chromogens that can be used in this manner include, but are not limitedto, guaiac, 2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid),tetramethylbenzidine, mixtures of phenol and 4-aminoantipyrine, and4,5-dihydroxy-naphthalene. Examples of redox catalysts are peroxidases,iron protoporphyrin and metal ions. Any amino acid capable of beingoxidized by oxygen in the presence of an amino acid oxidase to producehydrogen peroxide can be used. Examples are alanine, leucine, serine,phenylalanine, aspartic acid, and tyrosine.

Examples of substrates with reporter groups meeting the abovedescriptions arecarbobenzoxy-L-valine-L-arginine-4-methoxy-2-naphthylamine,carbobenzoxy-L-arginine-L-arginine-4-methoxy-2-naphthylamine,carbobenzoxy-L-arginine-L-arginine-L-arginine-4-methoxy-2-naphthylamine,carbobenzoxy-L-leucine-L-arginine-4-methoxy-2-naphthylamine,carbobenzoxy-L-valine-L-arginine-4-methoxy-2-naphthylamine, andD-valine-L-leucine-L-arginine-4-methoxy-2-naphthylamine. Among these,the most preferred arecarbobenzoxy-L-arginine-L-arginine-L-arginine-4-methoxy-2-naphthylamineand D-valine-L-leucine-L-arginine-4-methoxy-2-naphthylamine.

Reporter groups that produce a color change directly upon release from asubstrate residue include, but are not limited to, such species asindoxyl and pH indicators such as chlorophenol red andtetrabromophenolphthalein ethyl ester. Indoxyl, for example, forms acolorless conjugate when attached to a substrate residue, and yet uponrelease from the residue reacts with atmospheric oxygen to form anintense blue color (indigo). Chlorophenol turns red upon release from aresidue when the pH is above the transition point of chlorophenol red,and tetrabromophenolphthalein ethyl ester turns blue upon release from aresidue when the pH is above its transition point. Other examples willbe apparent to those with experience in the use of pH indicators. Thechoice of pH indicator will depend in part on the pH at which enzymeactivity is detected, i.e., for those embodiments in which a low pH isnecessary the appropriate pH indicators will be those whose transitionpoints are within the same low range as that in which the assay isconducted. For those embodiments in which the pH is not restricted to alow range, pH indicators with higher transition points can be used.

In embodiments of the invention that utilize size exclusion to achieveselective detection of trichomonal hydrolase activity, the removal ofparticulate matter, including non-specific enzymatically activeparticular matter, results in the removal of a major portion of theinterfering enzymatic activity in the fluid without eliminating thesoluble or non-particulate hydrolytic activity that is attributable tothe presence of T. vaginalis. The size threshold, i.e., the smallestparticles that are removed from the fluid by the size exclusion, ispreferably 20 microns, more preferably 10 microns in diameter, and mostpreferably 1 micron in diameter. The size exclusion thus preferablyresults in a fraction that is devoid of all particles greater than 20microns in diameter, more preferably devoid of all particles greaterthan 10 microns in diameter, and most preferably devoid of all particlesgreater than 1 micron in diameter. Size exclusion can be achieved byconventional methods, including centrifugation, filtration (bothpressure filtration and vacuum filtration), sedimentation, andprecipitation. A particularly convenient method of achieving sizeexclusion is by the use of a test device that includes a solidchemically inert porous material that serves as a filter medium,arranged in the device in such a manner that the liquid sample passeslaterally through the porous material before reaching the test zone inwhich the enzymatic activity of the sample is tested. The test devicecan thus be designed to include an application site designated for entryof the sample, a test site where enzymatic activity either occurs or isdetected, and a lateral flow path between the application site and thetest site for movement of the sample either by capillary action,pressure differential, gravity flow, or any other driving force, withthe porous material either filling or traversing the flow path. Oneparticularly convenient configuration of the porous material is acontinuous elongated strip arranged such that capillary action draws thesample lengthwise along the strip.

To further exclude enzymatic activity that is not attributable totrichomonal hydrolases or to other hydrolases to which the assay may bedirected, the assay can be performed in the presence of one or more of aseries of hydrolase inhibitors that are specifically chosen to isolatethe hydrolase of interest. In assays for the detection of trichomonalhydrolase activity, these inhibitors include antipain, chymostatin,trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, and variouspolypeptides and dipeptides including Lys-Pro-Gln-Leu-Trp-Pro,Arg-Lys-Asn-Val-Tyr, and Lys-Pro. In certain cases, the amount ofinhibitor present will affect its selectivity, i.e., its ability toinhibit the enzyme (including hydrolase) activity that is notattributable to trichomonal or other target hydrolases withoutinhibiting the target hydrolase activity. This is generally a matter ofdegree, since in some cases there may be inhibition of all hydrolaseactivity to some extent and to varying degrees from one hydrolase to thenext, and some inhibition of the target hydrolase activity can betolerated while still producing a viable assay. The amount of anyparticular inhibitor that will result in preferential inhibition of thenon-target hydrolase activity to an extent sufficient to produce areliable assay is readily determinable by routine experimentation wellwithin the routine skill of the laboratory technician. For selectiveinhibition of interfering, non trichomonal hydrolase activity, antipain,for example, is used at a preferred concentration range is 5micrograms/mL to about 40 micrograms/mL, and for chymostatin, apreferred concentration range is from about 0.2 mM to about 10 mM.

As in the embodiments of the invention that require a low pH, thesubstrate in embodiments that employ size exclusion and hydrolaseinhibitors can be a conjugate that consists of a reporter group bondedto a substrate residue by a covalent bond that is cleavable byhydrolysis, the reporter group being a species that produces adetectable change in an indicator upon contact with the indicator. Whena substrate of this type is used, the substrate and the indicator areplaced in individual, spatially separated regions in the test device,and the device is constructed to cause the sample to contact the tworegions in succession, ultimately placing the released reporter group incontact with the indicator. According to one such arrangement, theindicator region is placed upstream of the substrate region relative tothe direction of flow, and the indicator is soluble in the sample whilethe substrate is either fixed in position and poorly soluble in thesample or simply placed downstream such as at the end of the porousstrip that constitutes the test device. In this arrangement, only theindicator travels with the sample flow. In an alternative arrangement,the positions are reversed. In either arrangement, the indicator can beany species that produces a detectable signal, whether machine-readableor visually detectable, and whether directly or indirectly through other(intermediary) species. Preferred indicators are those that produce avisually detectable signal such as fluorescence, chemiluminescence, or asimple color change. The reagent is likewise any species that causes thechange in the indicator to occur. Examples of visually detectableindicators are p-dimethylaminocinnamaldehyde, diazonium dyes, andtetrazonium compounds, although many others are known to those skilledin the art and can be used in these assays. The individual indicatorslisted above are specific examples that can be used here as well.Preferred reporter groups are those that react with the indicator in themost intense and efficient manner. Examples of reporter groups usefulfor this purpose are 4-methoxy-2-naphthylamine, beta-naphthylamine, and7-amino-4-methylcoumarin, and analogs and derivatives of thesematerials, α-naphthol, β-naphthol, 3-hydroxy-2-naphthoic acid,6-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthalenesulfonic acid,1-naphthol-3,6-disulfonic acid, 6-bromo-2-naphthol, 6-hydroxy-2-naphthyldisulfide, and 4-hydroxy-1-naphthalenesulfonic acid.

Another type of substrate that can be used in these embodiments thatemploy size exclusion and selective hydrolase inhibitors is a conjugateof a chromogen bonded to a binding member by a covalent bond that iscleavable by hydrolysis, the chromogen being a species that itselfundergoes a detectable change upon release from the binding member. Thedescription of chromogens presented above in connection with embodimentsof the invention that focus on hydrolytic activity at low pH isapplicable to these embodiments as well.

Still further selectivity toward trichomonal hydrolase activity inembodiments of the invention that utilize size exclusion and one or moreof the inhibitors mentioned above can be achieved when the bindingmember of the conjugate described in the preceding paragraph is apeptide bonded to the reagent at the C-terminus of the peptide, witheither lysine or arginine as the amino acid at the C-terminus. Preferredpeptides are those of 1 to 6 amino acids, while the most preferred arethose with 2 to 3 amino acids. It is also preferred that the N-terminusof the peptide be protected against hydrolysis by an N-blocking group,examples of which are cited above.

In certain implementations of the invention, the performance of the testwill benefit by diluting the sample of bodily fluid with an aqueousdiluent before applying the sample to the test device. Dilution of thesample can lower the viscosity of the sample to improve its flowcharacteristics. The diluent can also provide a means of controlling thepH of the sample in cases where the test results may vary with thesample pH. Thus, the preferred diluents in many cases are aqueoussolutions of buffering agents, particularly those that stabilize thediluted sample at a pH of from about 6.5 to about 7.5. One suchbuffering agent is imidazole; others will be readily apparent to thoseskilled in the art.

While the descriptions above are directed primarily to assays of vaginalfluid specimens, the methodologies of this invention are useful in thedetection of a variety of hydrolases or other target enzymes that can bedetected in different bodily fluids, such as blood, urine, saliva, andcerebrospinal fluid. Assays for Trichomonas vaginalis are best conductedon vaginal fluid specimens.

Test devices are readily designed to contain the features thatcharacterize each the assays and embodiments described above. Thepreferred test devices are those that contain all components of theassay in solid dry form, requiring only the application of a specimen ofbodily fluid and at most a minimal number of additional reagents orcomponents. In particularly preferred embodiments, the test device isentirely self-contained, requiring only the application of the specimen,either at full strength or diluted with an aqueous buffer solution.Application of the specimen can achieved by an appropriate transferimplement, such as a swab, a finger cot, a cervical brush, a dropper, ora pipette or other type of aspiration tube, or generally any device thatcan be used to collect and transfer the specimen, or by methods such asa vaginal wash. Swabs may consist of cotton, Dacron, or any othernatural or synthetic absorbent material affixed to a handle made ofplastic, wood, or other rigid material.

The test device will be designed to allow detection of the change thatindicates the presence of hydrolase activity as an immediate or quickresponse to the application of the specimen. In some cases thedetectable change can be read or observed, and quantitated if desired,on the implement used to apply the sample, while in others the changecan be read, observed, or quantitated on the test device. Depending onthe particular test, the device may contain a built-in acidifying agentor a filtering material, and assay components deposited in strips, zonesor other regions of defined dimensions. Those components that must bekept separate until the sample is added are isolated by gaps or barriersthat provide the spatial separation. In many cases, some of the assaycomponents will be affixed to the inner surface of the device by bindingmatrices that are insoluble in the sample liquid while others aresoluble and readily dispersible in the sample liquid upon contact totravel with the sample through the device.

As noted above, assays in accordance with this invention can beperformed with swabs and other transfer implements that pick up theassay components and permit the user to read the assay result directlyon the implement. Transfer implements can be used with both types ofsubstrates described above, i.e., those whose reporter groups arechromogens or other species that undergo a detectable change as a directresult of their release from the substrate residue, and those whosereporter groups are reagents that produce a detectable change in aseparate indicator. When reporter groups that produce a change in aseparate indicator are used, the implement can first be applied to thesubstrate and then to the indicator. In some cases, a proper result canbe achieved by contacting the indicator within thirty seconds ofcontacting the substrate. In others, best results will be achieved whenthere is more of a time delay between these two contacts to allow theenzyme time to act upon the substrate and to avoid or minimize anyinhibitory effect the indicator may have on the enzyme. In these cases,preferred time delays are at least two minutes, and most preferably fromabout five minutes to about thirty minutes.

In test devices where the solid-phase reagents reside in films on theinner surface of the device, these films may be formed by applying thereagents in liquid form followed by drying or other solidification. Theliquid form of the reagent can for example be a solution or suspensionof the reagent, or an uncured liquid form of a support matrix in whichthe reagent will be retained. The solidification step can thus be anevaporation of the solvent or suspending liquid or a curing of thematrix precursor. Additional materials may be included in the film for avariety of purposes, such as for example:

-   -   (1) to facilitate the application of the liquid to the surface        by modifying the viscosity of the liquid,    -   (2) to help form a continuous smooth solid layer that remains        uniform and does not disintegrate or granulate over time or upon        the application of additional layers over the initial layer, or    -   (3) to modify the solubility of the layer with solvents used in        layers to be applied over the layer or to make the layer soluble        in solvents which do not dissolve layers applied underneath; or        combinations of these purposes. Polymeric materials can be used        for one or all of these purposes. Celluloses and various        cellulosic derivatives are particularly useful, the derivatives        substituted appropriately to achieve the desired solubility        characteristics.

The reagent films in these test devices may also include additives thatenhance stability and shelf-life as well as additives that facilitatedissolution of the substrate, the catalytically active hydrolase, orother components that function best when in solution. Examples ofadditives that improve the stability of the reagents are buffers;antioxidants such as butylated hydroxytoluene (BHT), butylatedhydroxyanisole (BHA), ascorbate, and dithiothreitol (DTT); as well asmetal binding components and chelators such asethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis(β-aminoethyl ether) (EGTA). Examples of additives thatfacilitate the dissolution, solubilization, or dispersion of the variouscomponents are mannitol, sorbitol, polyethylene glycol, lactose, andmild detergents. Detergents or other lysing agents can also be includedfor purposes of lysing the trichomonads to release internal hydrolasesfrom the trichomonads into the liquid.

In addition to the zones of assay components described above, preferredtest devices of this invention include a built-in positive control, andfurther preferred devices include both a built-in positive control and abuilt-in negative control. The positive control is defined as asubstance or region on the device that causes the device to emit thesame detectable signal that would be caused by application of an actualspecimen that displays the target hydrolase activity, or a similardetectable signal, except that the positive control produces the signalupon the application of any specimen or substitute fluid regardless ofwhether or not the specimen or substitute fluid displays such activity.The positive control thus indicates to the user that the detectablesignal itself is functioning properly and is capable of being generated,thereby assuring that false negative readings will not occur when actualspecimens are applied. A preferred positive control is one that isformed by an additional reagent zone located on the device separatelyfrom the actual test (substrate and indicator) zones. This positivecontrol can consist of any chemical that would react with the indicatorto produce a detectable response, such as one of the reporter groupsincorporated in peptide substrates described above, or an analog of thereporter group. A preferred positive control material is sodiumaminonaphthoate, which reacts with an indicator to produce a detectableresponse much as the reporter groups listed above, but possessessuperior stability and extended shelf-life.

A negative control is a control that indicates to the user that the testdevice will not produce a signal when the applied sample is devoid ofthe target hydrolase activity. The negative control thereby assures theuser that the device will not produce false positive results when anactual specimen is applied. The negative control can be activated by thesame bodily fluid on which the test for the target hydrolase activity isperformed or by a separate specimen of the fluid than the specimen usedfor the test, or by a substitute fluid such as the diluent used with thetest specimen. One type of negative control is a zone that contains thesame indicator as in the indicator test zone, but is located on thedevice at a location separate from the substrate zone, indicator testzone, and the positive control zone. When a specimen is applied to thisnegative control zone without having first been applied to thesubstrate, the zone is checked to see whether a change is observed inthe indicator that is located in that zone. Such a change would indicatethat the specimen contains an interferent, other than the targethydrolase activity, that directly causes the change in the indicator.One example of such an interferent is resorcinol that is present in somevaginal products and that by itself produces a color change in diazoniumsalts. Another type of negative control is one that includes both theindicator and an inhibitor of the target hydrolase. When a specimen isfirst applied to the substrate and then to this negative control zone,any color formation or other signal generation in this the negativecontrol zone would then indicate the presence of an interferent in thespecimen that causes the change in the indicator and is not affected bythe inhibitor, i.e., an interferent other than the target hydrolase thatcauses the indicator to change.

According to one method of performing a negative control test in whichthe detectable signal is read on the transfer implement itself, the userplaces one or two drops of diluent on either the negative controlreagent zone or on a clean implement, and then rubs the implement on thenegative control zone. If no detectable response is seen on theimplement, the negative control test indicates that no false positiveresults will occur when an actual specimen is applied to the test zone.If the implement develops a detectable response, the negative controltest indicates that there is a risk of a false positive result when anactual specimen is applied to the test zone. After performing asuccessful negative control test, the user can then rub the sameimplement on the positive control zone to perform the positive controltest. If the implement then fails to produce a detectable response, thepositive control test would be interpreted as failed, i.e., there is arisk of false negative results when test specimens are applied. Ifhowever the implement develops a detectable response in the positivecontrol test, the positive control test has succeeded, indicating thatfalse negative results will not occur because of device failure. Thepositive and negative control tests are designed primarily to check thecondition of the indicator, which in many cases will be the least stablereagent in this type of test device. These control tests can also beused to detect the presence of interferents in a specimen.

An alternative positive control test is one that tests the condition ofthe indicator while also testing for interfering substances in the testspecimen. This control test can be performed using the implementcontaining the actual specimen to be tested for trichomonal hydrolaseactivity, rather than a separate implement. First, the diagnostic testfor trichomonal hydrolase activity is performed. If the diagnostic testresult is positive, there is no need for a positive control test, sincethe diagnostic test itself will have established that the indicator isfunctional. If the diagnostic test result is negative, the positivecontrol test should be performed immediately afterward by rubbing thesame specimen-containing implement on the positive control reagent zone.If a detectable response is produced at the positive control zone, thepositive control test and hence the diagnostic test are interpreted asvalid. If no detectable positive control response is produced, i.e., theresult is negative rather than positive, the positive control isinterpreted as failed, and the preceding diagnostic test would beconsidered invalid. A second fluid specimen from the same individual canalso be used to perform the negative control to detect positiveinterference from the specimen itself. If the negative control testproduces no detectable signal, the negative control test and hence theactual diagnostic test are interpreted as valid. Alternatively, if thenegative control test produces a detectable signal upon exposure to thefluid sample, i.e., the control test is positive instead of negative,the negative control is interpreted as failed and the precedingdiagnostic test would be considered invalid.

A still further alternative for a positive control test is one that usesa control hydrolase in place of the sodium aminonaphthoic acid or otherreagent that reacts directly with the indicator. This type of positivecontrol tests the functionality of the substrate conjugate, indicatorand reaction conditions. As with the positive controls described above,a control hydrolase resides in a separate zone, which the user rubs withan implement immediately after a negative test result is obtained fromthe diagnostic test. The results are then interpreted in the same way asin the preceding control test, but only after sufficient time haselapsed for the hydrolase to generate a positive result. The length ofthe additional waiting period depends on the nature and concentration ofthe hydrolase used. The hydrolase must be active on the substrate at thepH used in the device. Accordingly for a low pH test, a preferredhydrolase for the positive control zone is bromelain, an inexpensivethiol hydrolase that is stable in dry form and active at a low pH.Bromelain can be deposited in a dry film in a positive control reagentzone in sufficient quantity to provide a positive control result in lessthan two minutes.

To summarize, the functions that can be served by positive and negativecontrols are as follows:

(a) A positive control can provide assurance to the user that all testelements (the substrate conjugate, the indicator and the reactionconditions) are performing correctly, and can be relied upon to detecttrichomonal or other target hydrolase activity in a specimen, if suchactivity is indeed present. A negative control can provide assurancethat the test reagents can be relied upon not to generate a positiveresult in the absence of trichomonal or other target hydrolase activity.By performing these functions, the positive and negative controls serveas means for checking the quality of the reagents in the test device.

(b) A positive control can provide assurance that the specimen beingtested does not contain interferents that can inhibit the trichomonal orother target hydrolases or, by other means, interfere with the abilityof the trichomonal or other target hydrolases to generate a positivetest result. Similarly, a negative control can provide assurance thatthe specimen being tested does not contain interferents that cangenerate a positive response in the absence of the hydrolase. Byperforming these functions, the positive and negative controls serve asmeans for checking the quality of the specimen itself.

(c) If a test device and its controls are designed and constructed suchthat the control elements of the device are the last portions of thetest device to be contacted by the specimen either because of theirlocation on the test device or because of the protocol for applying thespecimen to the device, a properly functioning positive control providesan indication that the test device has been filled, or properly wetted,with the specimen. In serving this function, therefore, the positivecontrol serves as a means to check the test procedure. Controls of thistype are often termed “procedural controls.” Checking the procedure isparticularly important with clear or colorless specimens, in devicesdesigned to contain small volumes, and in devices in which specimen flowpaths are partially obstructed from view.

One example of a test device embodying some of the concepts of thisinvention is shown in FIG. 1. This device and others within the scope ofthis invention can be of any size and shape, but it is particularlyconvenient to use a device that is similar in size and shape to a commoncredit card. The view in FIG. 1 is a longitudinal vertical cross sectionwith the device held horizontally with its operative surface facingupward, this being the position in which the device is most likely to beused.

The base of the device is a solid rigid sheet 11 of inert non-porousmaterial to whose upper surface is affixed a strip 12 of cellulosic orsimilarly porous material. The strip 12 is affixed to the underlyingsheet by adhesive or any method that provides securement of the strip.The porous strip has two reagent zones 13, 14 defined at discreteregions along its length, each reagent zone having been applied bypipetting, stenciling, painting, printing or spraying techniques or anyother method of deposition. Also shown is a swab 15 that is first wettedwith a specimen of vaginal fluid and then applied to the test device atone end of the porous strip 12 to transfer the specimen to the porousstrip. As noted above, the specimen can be applied to the porous stripby any implement capable of transferring a specimen; a swab is but oneexample. Once the specimen is applied, the specimen travels the lengthof the porous strip (left to right in the view shown in these Figures)by capillary action through the strip.

The first reagent zone 13 encountered by the migrating specimen is onethat contains an indicator, and in those tests that involve aninhibitor, both the indicator and inhibitor are present in the firstreagent zone as a solid mixture. The second reagent zone 14 contains asubstrate that is susceptible to trichomonal hydrolytic activity. Theindicator, and the inhibitor when present, are preferably deposited inthe first reagent zone 13 in a manner permitting them to disperse in thespecimen as the specimen migrates past, the specimen thereby drawing theindicator and inhibitor with it such that all three travel togethertoward the second reagent zone 14. The substrate in the second reagentzone 14 is preferably a conjugate consisting of a binding membercovalently bonded to a reagent that causes a detectable change in theindicator. In alternative but similar devices, the relative positions ofthe two reagent zones can be reversed.

The device of FIG. 2 is a variation of the device of FIG. 1, designedfor applications where a separate development reagent is used forenhancing the detectability of the change in the indicator. The swab ofFIG. 1, although not shown, is used to apply a specimen to this testdevice in the same manner as in FIG. 1, and the test devices areidentical except that the test device of FIG. 2 contains a second sheetof solid non-porous inert support material 16 affixed to the first sheetby means of adhesive to form a flap that extends over the second reagentzone 14. On the side of the flap that faces the lower sheet is a layerof the development reagent 17 normally separated from the second reagentzone 14 by a gap 18. Once the specimen has reached the second reagentzone 14, the flap is pressed against the lower portion of the device toplace the development reagent in contact with the second reagent zone.The flap is then lifted and the second reagent zone is observed todetermine whether the detectable change has occurred.

A further variation is the device shown in FIGS. 3 a, 3 b, 3 c, and 3 d.This device is similar to that of FIG. 2, except that the flap 16 isseparated from the reagent zone 14 on the lower portion (or base sheet)of the device by an intermediate sheet or shield 20. This intermediatesheet 20 is at least partially transparent. Application of the specimen,as shown in FIG. 3 b, is achieved by inserting a wetted swab (or otherimplement carrying the specimen) between the base sheet 11 and theintermediate sheet 20. The intermediate sheet 20 is bonded or otherwisesecured to the base sheet 11 along the longitudinal edges of thesesheets, which are not visible in these Figures since the Figures arelongitudinal vertical cross sections. The left ends of the sheets arenot bonded together but instead left open to form an opening forinsertion of the swab 15, as shown in FIGS. 3 b, 3 c, and 3 d. The swabis inserted deeply enough that its head comes into contact with thenearest end of the porous strip 12, and migration of the specimenproceeds as in the devices of the preceding Figures. After sufficienttime has elapsed to allow the specimen to reach the second reagent zone14 (which contains the substrate), the device is manually bent as shownin FIG. 3 c, causing the flap 16 to separate from the base sheet 11 andthe intermediate sheet 20, and due to the resiliency of the intermediatesheet 20 thereby causing the intermediate sheet 20 to lift off of (andout of contact with) the base sheet 11. The device is then allowed toresume its flat configuration (FIG. 3 d), which may occur by simplyreleasing the manual pressure on the device provided that the device issufficiently resilient. Upon resuming this configuration, the flap 16comes into direct contact with the porous sheet 12, thereby bringing thedevelopment reagent 17 in contact with the indicator zone 14.

The various sheets shown in FIGS. 1, 2, and 3 a through 3 d arefabricated of solid material that is chemically inert to all of thespecimens, reagents, diluents and other materials that contact thesesheets during the assay, and the sheets are preferably semi-rigid with aresiliency that allows them to be distorted (for example as shown inFIG. 3 c) but causes them to resume their shape when they are releasedfrom the distortion. The reagent zones can be formed by applying thereagents to specific areas on any of the sheets by conventional methodsof deposition such as printing, bonding, or the application of foils,paper disks, or the like. Indicia can also be placed on the sheets inthe same manner. The intermediate sheet 20 and the flap 16 can betransparent to permit viewing of the regions that show the test results,or the ability to observe the results can be achieved by strategicallyplaced holes that are either stamped, punched, or cut out ofnon-transparent sheets. One or more of the sheets can be opaque, sincethe test results will in most cases be viewed from only one side of thedevice. When transparent materials are used, suitable examples arepolyethylene terephthalates (such as, for example, MYLAR®). The porousstrip 12 can be made of a cellulosic or synthetic material that isreadily wetted by aqueous liquids, that possesses the desired wickingand particle retention properties, and does not strongly bind or adsorbproteins. Examples of suitable materials are Ahlstrom 237 Grade filterpaper and Whatman Grand 44 filter paper. The reagent zones can beapplied to the porous strip as dried deposits or coatings, in geometricor specially arranged patterns that avoid direct contact between thereagents in different zones until the strip is wetted.

The test devices that contain two or three sheets can be formed in avariety of ways. Preferred methods involve securing together orlaminating sheets of polymeric material with adhesives or heat sealing.The laminated sheets can form a thin, flattened card, or one or more ofthe sheets can be pressure-formed to form an open pocket (3 a through 3d) for ease of insertion of the swab. The pocket is simply a conveniencemeans to hold the swab against the end of the porous strip. Alternativemeans are readily devised.

A still further type of test apparatus embodying the concepts of thisinvention is shown in FIG. 4. This apparatus consists of a single sheet30 with distinct reagent zones 31, 32, 33, 34 on its surface, and a swab35. This apparatus is used by simply wetting an implement (shown in theFigure as a swab 35) with the specimen, then rubbing the wettedimplement over the various reagent zones in a preselected sequence, andfinally reading the test result on the implement itself. In thearrangement shown in FIG. 4, the leftmost reagent zone 31 contains asubstrate, and the reagent zone 32 to the right of the substrate zonecontains an indicator, both the substrate and indicator as describedabove. Optionally, one or more inhibitors of non target hydrolases canbe incorporated into reagent zone 31. Rubbing the wetted implement firstover the substrate zone 31 will cause substrate (and inhibitor, ifpresent) to adhere to the swab, and then rubbing the implement over theindicator zone 32 will cause the indicator to adhere to the implement aswell. The time interval between rubbing the implement over the substratezone and then over the indicator zone can be varied to meet the specificneeds of different target hydrolases. As in the other test devices andin the general description above, if the specimen contains trichomonalor other target hydrolase activity, this activity will cause hydrolyticcleavage of the substrate which will in turn result in a visible changein the indicator that is adhering to, or has otherwise been picked upby, the implement. The remaining zones are a negative control zone 33and a positive control zone 34, both of which are used by rubbing theimplement (independently of the indicator and substrate conjugate zonesand of each other) with a specimen-wetted implement (or an implementwetted with a fluid other than a specimen) and determining that no colorchange (or other detectable change) appears on the implement for thenegative control and that a color change (or other detectable change)does appear for the positive control, both of which are independent ofwhether or not there is trichomonal hydrolase activity in the specimen.

The following examples are offered for purposes of illustration only.Amino acids are represented by their common three-letter or one-lettercodes, and the following additional abbreviations are used: CBZ or Zcarbobenzoxy BZ benzoyl BOC tert-butoxycarbonyl MNA4-methoxy-2-naphthylamine βNA β-naphthylamine AMC7-amido-4-methylcoumarinPreparation of MaterialsA. Trichomonal Hydrolases

MATERIALS: T. vaginalis culture (ATCC 30001) and collection bufferconsisting of 14 mM maltose, 6 mM L-cysteine and 10 mM HEPES inphosphate buffered saline, approximately pH 6.5.

PROCEDURE: Hydrolases secreted by live trichomonads were collected bywashing trichomonads in late log-phase culture by centrifugation once incollection buffer, resuspending the trichomonads at 10⁷ organisms/mL incollection buffer, incubating four hours at 35° C., pelleting bycentrifugation, and filtering through a 0.45 micron pore filter.Aliquots of the filtrate were placed in microfuge tubes and stored at−80° C. until use.

B. Concentrated Trichomonal Hydrolases

The materials and procedure were identical to those used in thepreparation of trichomonal hydrolases, but with the organisms at tentimes the density. Trichomonads in late log-phase culture were washed bycentrifugation once in collection buffer, resuspended at 10⁸organisms/mL in collection buffer, incubated 4-5 hours at 35° C., andpelleted by centrifugation. Aliquots of the supernatant were placed inmicrofuge tubes and stored at −80° C. until use.

C. Soluble Trichomonal Hydrolases from Lysed T. vaginalis Cells

MATERIALS: T. vaginalis culture (ATCC 30001) and wash buffer consistingof 14 mM maltose in phosphate buffered saline, approximately pH 6.5.

PROCEDURE: Hydrolases were collected from lysed trichomonads by washingthe trichomonads in late log-phase culture by centrifugation once inchilled (4° C.) wash buffer, resuspending at 10⁸ organisms/mL indeionized water, holding for one hour at room temperature to allow theorganisms to swell up, vortexing two minutes to lyse the organisms, andpelleting by centrifugation. Aliquots of the supernatant was then placedin microfuge tubes and stored at −80° C. until use.

D. Total (Soluble and Particulate) Trichomonal Hydrolases from Lysed T.vaginalis Cells

Using the same materials as those of C above, a similar procedure wasused, except that hydrolases were collected from a different isolate ofT. vaginalis, and the whole lysate was used rather than only thesupernatant. Trichomonads in late log-phase culture were washed bycentrifugation once in chilled (4° C.) wash buffer, resuspended at 10⁸organisms/mL in deionized water, held 3-4 hours at 4° C. to allow theorganisms to swell up, and vortexed briefly to lyse the organisms, andaliquots of the suspension were placed in microfuge tubes and stored at−80° C. until use.

E. ZRRR-MNA Substrate Films

MATERIALS: CBZ-Arg-Arg-Arg-MNA triacetate (ZRRR-MNA), 200 mM L-cysteinehydrochloride in water, and 25% (w/w) hydroxypropylcellulose in ethanol.

PROCEDURE: A 400 mM ZRRR-MNA stock solution was made by dissolvingZRRR-MNA in ethanol. For a solution containing 200 mM ZRRR-MNA, 20 mML-cysteine, and 5% hydroxypropylcellulose, a mixture was made of 100 μL400 mM ZRRR-MNA, 20 μL 200 mM L-cysteine, 40 μL ethanol, and 40 μLhydroxypropylcellulose in ethanol. For solutions containing lowerconcentrations of ZRRR-MNA, the volume of ZRRR-MNA was decreasedcorrespondingly, and the difference in volume made up by addingadditional ethanol. Substrate films were made by pipetting 1 μL of themixture over a circular area approximately ¼-inch (0.64 cm) in diameteron a sheet of Mylar, and drying under a stream of dry nitrogen. Thedried films were stored in a container with desiccant until used.

F. VLR-MNA Substrate Films

MATERIALS: D-Val-Leu-Arg-MNA (VLR-MNA), 200 mM L-cysteine hydrochloridein water, and 25% (w/w) hydroxypropylcellulose in ethanol.

PROCEDURE: A 400 mM VLR-MNA stock solution was made by dissolvingVLR-MNA in a mixture of 80% (v/v) ethanol and 20% (v/v) 3M hydrochloricacid. For a film made using a 200 mM VLR-MNA solution, a mixture wasmade of 100 μL 400 mM VLR-MNA, 20 μL 200 mM L-cysteine, 64 μL ethanol,and 16 μL hydroxypropylcellulose in ethanol. Substrate films were madeby pipetting 1 μL of the mixture over a circular area approximately¼-inch (0.64 cm) in diameter on a sheet of Mylar, and drying under astream of dry nitrogen. The dried films were stored in a container withdesiccant until used.

G. Indicator Films Containing Diazonium Dyes

MATERIALS: Fast Red RL or Fast Garnet GBC, 10% w/w ethylcellulose inmethanol, and Meyer rod, rubber roller, or other device for applying athin film of liquid onto a plastic sheet.

PROCEDURES: Fast Red RL or Fast Garnet was dissolved indimethylformamide, diluted in methanol, and then mixed with an equalvolume of 10% ethylcellulose to achieve a final solution containing 10%v/v dimethylformamide, 5% w/w ethylcellulose, and the desiredconcentration of diazonium dye (10 mM Fast Garnet or 64-100 mM Fast RedRL). The dye solution was applied to a Mylar sheet as a thin coatingusing a #10 Meyer rod, roller or other means, and subjected to a flow ofwarm air for rapid drying. The dried films were stored in a containerwith desiccant until used.

Experiment 1

This experiment examines the hydrolysis of the substratesCBZ-Arg-Arg-MNA (ZRR-MNA) and D-Val-Leu-Lys-MNA (VLK-MNA) by trichomonalhydrolases over the pH range 2.8 to 9.2 and examines the impact ofvaginal fluid on hydrolase activity at each pH.

Materials

-   -   200 mM buffer solutions:        -   Glycyl-glycine (Gly-gly), pH 2.8        -   Sodium acetate, pH 5.1        -   2-(N-Morpholino)propane-sulfonic acid (MOPS), pH 7.0        -   N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),            pH 7.5        -   Tris(hydroxymethyl)aminomethane (TRIS), pH 8.0        -   Sodium borate, pH 9.5    -   Pooled normal vaginal fluid supernatant (NVS), prepared as        follows: vaginal fluid specimens collected on Dacron swabs from        normal, uninfected women were centrifuged to extract the fluid        from the swabs, and the pooled fluid was frozen until use. The        thawed fluid was centrifuged to pellet the particulate matter,        the particulate materials and the supernatant were separated and        each was diluted 1:10 in acetate buffer.    -   2 mM CBZ-Arg-Arg-MNA acetate (ZRR-MNA), first prepared as a 100        mM solution in dimethylformamide, then diluted to 2 mM in        deionized water    -   2 mM D-Val-Leu-Lys-MNA (VLK-MNA), first prepared as a 100 mM        solution in dimethylformamide, then diluted to 2 mM in deionized        water    -   Indicator solution consisting of a freshly-made mixture of 100        μL of 25 mM para-dimethylamino cinnamaldehyde in ethanol, 900 μL        of 10% w/v 2% sodium dodecyl sulfate, 1.35 mL of 100 mM citric        acid, 450 μL of 200 mM disodium phosphate, and 3.5 mL water    -   100 mM acetic acid buffer, pH 3.7        Procedures

Assays without normal vaginal fluid supernatant present: Two sets of sixwells in a microtiter plate were prepared, with each well containing 80μL of one of the six 200 mM buffers, 10 μL of water and 10 μL of dilutedtrichomonal hydrolases. Ten μL of ZRR-MNA was added to each well in thefirst set of wells, and 10 μl of VLK-MNA was added to each well in thesecond set of wells. After fifteen minutes, 100 μl of indicator solutionwas added to each well. Because a high pH can reduce the intensity ofcolor produced by the indicator solution, 50 μl of 100 mM acetic acidbuffer, pH 3.7, was added to each well except the pH 2.8 wells (waterwas substituted for the acetic acid in those wells). After anotherfifteen minutes, the absorbance of the solutions in the wells wasmeasured on a microtiter plate reader set on 540 nm wavelength.

Assays with normal vaginal fluid supernatant (NVS) present: Theprocedure of the preceding paragraph was followed, except that 10 μL ofdiluted NVS was added to the wells in place of the 10 μL of water.

Results

Assays without normal vaginal fluid supernatant present: The hydrolyticactivity of the trichomonal enzymes, as reflected by the development ofa pink or fuchsia color and increased absorbance at 540 nm, was highacross a very broad pH range, as shown in Table 1.1 below. When ZRR-MNAwas used as the substrate, the enzyme activity varied only slightlybetween pH 2.8 and pH 8.0 (absorbance readings ranged from 0.631 to0.738), with a decrease observed only when the pH was 9.2 (absorbancedropped to 0.172). A different pH profile was observed when VLK-MNA wasused as the substrate, with a peak of activity at pH 5.1 (absorbance of1.622) and high activity at pH 9.2 (absorbance of 1.459). Across theentire pH range tested, a more intense color was produced with VLK-MNAcompared to ZRR-MNA. TABLE 1.1 Assays Without Vaginal Fluid SupernatantPresent: Absorbance Readings at Different pH Levels Buffer: Gly-glyAcetate MOPS HEPES TRIS Borate pH: Substrate pH 2.8 pH 5.1 pH 7.0 pH 7.5pH 8.0 pH 9.2 ZRR-MNA 0.631 0.792 0.638 0.738 0.667 0.172 VLK-MNA 0.6911.622 1.087 1.217 1.219 1.459

Assays with normal vaginal fluid supernatant (NVS) present: The presenceof a small amount of vaginal fluid greatly reduced the hydrolyticactivity of the trichomonal hydrolases, as shown in Table 1.2 below.Hydrolysis of ZRR-MNA in the presence of NVS was low at pH 2.8, 5.1, and9.2 (absorbance readings of 0.082-0.089), and hydrolysis of VLK-MNA inthe presence of NVS was low across the entire pH range tested(absorbance readings of 0.062-0.094). With ZRR-MNA as the substrate,enzyme activity was highest in pH 8 TRIS buffer (absorbance reading of0.403); at this pH, the color production was 60% of that observed whenvaginal fluid was not present (0.403/0.667). TABLE 1.2 Assays WithVaginal Fluid Supernatant Present: Absorbance Readings at Different pHLevels Buffer: Gly-gly Acetate MOPS HEPES TRIS  Borate pH: Substrate pH2.8 pH 5.1 pH 7.0 pH 7.5 pH 8.0 pH 9.2 ZRR-MNA 0.085 0.082 0.313 0.3860.403 0.089 VLK-MNA 0.094 0.065 0.066 0.063 0.073 0.062Interpretation

These results show that trichomonal hydrolases are active across a broadpH range, but that the optimum pH for maximum hydrolase activity varieswith the substrate used. The optimum pH is also affected by the presenceof vaginal fluid supernatant, which can be strongly inhibitory. Forexample, when vaginal fluid is not present in the assay mixture, theproduction of pink color from hydrolysis of ZRR-MNA by trichomonalenzymes is similar whether the pH is as low as 2.8 or as high as 8, butin the presence of vaginal fluid, color production is much lessinhibited at pH 7 or 8 than at pH 2.8 or 5.1. The presence of vaginalfluid supernatant in the assay can strongly affect whether a specificsubstrate is readily hydrolyzed by trichomonal enzymes. This isespecially true for VLK-MNA, which is readily hydrolyzed by trichomonalenzymes when no vaginal fluid is present, but poorly hydrolyzed in thepresence of even a small amount of vaginal fluid. To produce a usefultest system based on the activity of trichomonal hydrolases in thepresence of vaginal fluid, it is important to define the best substrateand pH using either a mixture of trichomonal hydrolases and normalvaginal fluid supernatant prepared in the laboratory, or vaginal fluidspecimens from subjects with trichomoniasis. For the initial selectionof suitable substrates that are readily hydrolyzed by trichomonalhydrolases, vaginal fluid supernatant, rather than whole vaginal fluid,is preferred, since whole vaginal fluid contains non-trichomonalhydrolases associated with the particulate matter (as the experimentsbelow will demonstrate).

Experiment 2

This experiment examines the hydrolysis of the substratesCBZ-Arg-Arg-MNA (ZRR-MNA), BOC-Leu-Arg-Arg-AMC (BLRR-AMC),BOC-Leu-Lys-Arg-AMC (BLKR-AMC), and CBZ-Lys-Lys-Arg-AMC (ZKKR-AMC) bytrichomonal hydrolases and vaginal fluid hydrolases.

Materials

-   -   Buffer solutions consisting of 200 mM sodium acetate, pH 5, or        200 M Tris-(hydroxymethyl)aminomethane (TRIS), pH 8    -   100 mM CBZ-Arg-Arg-MNA acetate (ZRR-MNA), in ethanol    -   100 mM BOC-Leu-Arg-Arg-AMC (BLRR-AMC) in dimethylformamide    -   100 mM BOC-Leu-Lys-Arg-AMC (BLKR-AMC) in dimethylformamide    -   100 mM CBZ-Lys-Lys-Arg-MNA (ZKKR-AMC), in water    -   6.3 mM L-cysteine hydrochloride in water    -   pooled normal vaginal fluid supernatant (NVS) prepared by        collecting vaginal fluid specimens on Dacron swabs from normal,        uninfected women, centrifuging to extract the undiluted fluid        from the swabs and to pellet the particulate matter, and pooling        the supernatants    -   whole normal vaginal fluid (NVF) from a single normal,        uninfected donor, prepared by centrifuging a Dacron swab        containing a vaginal fluid specimen to extract the undiluted        fluid from the swab, following by mixing to resuspend the        particulate matter in the fluid    -   96-well microtiter plate with 200 μL microwells.    -   60-well microwell mini tray with 20 μL conical microwells.    -   indicator solution consisting of 0.4 mM para-dimethylamino        cinnamaldehyde, 1.4% (w/v) sodium dodecyl sulfate, and 100 mM        citric acid (pH 4).        Procedure

Substrate solutions containing 8 mM of one of the four substrates(ZRR-MNA, BLRR-AMC, BLKR-AMC, ZKKR-AMC) in one of the two buffers(acetate, TRIS) were prepared by mixing 4 μL 100 mM substrate with 6 μL6.3 mM L-cysteine, 14 μL 200 mM buffer and 26 μL water. Assays oftrichomonal hydrolase activity, with or without the addition of NVS,were performed in 200 μL microwells. In one set of microwells, 10 μL ofeach substrate/buffer combination was mixed with 10 μL of undilutedtrichomonal hydrolases. In a second set of microwells, 10 μL of eachsubstrate/buffer combination was mixed with 10 μL of a mixture of 90%v/v trichomonal hydrolases and 10% NVS. Fifteen minutes later, 50 μL ofindicator solution was added to each well. After another fifteenminutes, the absorbance of each solution in the wells was measured on amicrotiter plate reader set on 540 nm wavelength.

Due to the partial opacity of cells and other particulate matter inwhole vaginal fluid, assays of hydrolase activity present in wholevaginal fluid were performed in 20 μL conical microwells and scoredvisually. The vaginal fluid from a single normal, uninfected donor wasagitated to resuspend the particulate matter, and 2 μL aliquots of thevaginal fluid were mixed with 2 μL of each substrate/buffer combination.To retard evaporation from the wells, water was pipetted into themini-tray adjacent to the wells prior to replacing the tray cover.Fifteen minutes later, 2 μL of indicator solution was added to eachwell. After another fifteen minutes, the color in each well was visuallyscored in 0.5-unit increments using the 0-6 scale presented in Table2.1. TABLE 2.1 Color Scale Color Color Score Yellow 0 Peach 1 Orange 2Light to medium pink 3 Dark pink or red 4 Fuchsia 5 Purple 6Results

Hydrolase activity by trichomonal hydrolases was detected with all fourof the peptide substrates tested, at both pH 5 (Table 2.2 below) and pH8 (Table 2.3 below). At pH 5, all four substrates registered sharpdecreases in activity when vaginal fluid was added. Inhibition oftrichomonal hydrolases from vaginal fluid supernatant was greatest withZRR-MNA, with a drop in absorbance from 0.420 to 0.068 at pH 5, and from0.385 to 0.182 at pH 8. No inhibition of trichomonal hydrolases fromvaginal fluid supernatant was observed with BLRR-AMC, BLKR-AMC, orZKKR-AMC at pH 8. Considerable hydrolytic activity was detected in wholevaginal fluid (with the particulate matter included) using ZRR-MNA at pH5, or any of the substrates at pH 8. However, BLRR-AMC, BLKR-AMC, andZKKR-AMC had comparatively low sensitivity to the hydrolases present inwhole normal vaginal fluid at pH 5. TABLE 2.2 Hydrolase Assays at pH 5Color Scores Substrate: ZRR- BLRR- BLKR- ZKKR- MNA AMC AMC AMCTrichomonal hydrolases 0.420 0.536 0.638 0.225 Trichomonal hydrolases +NVS 0.068 0.114 0.123 0.099 Whole normal vaginal fluid 3 0.5 0.5 0.5

TABLE 2.3 Hydrolase Assays at pH 8 Color Scores Substrate: ZRR- BLRR-BLKR- ZKKR- MNA AMC AMC AMC Trichomonal hydrolases 0.385 0.463 0.6490.357 Trichomonal hydrolases + NVS 0.182 0.568 0.731 0.352 Whole normalvaginal fluid 4 6 6 2Interpretation

While Experiment 1 demonstrated that trichomonal hydrolases could cleavepeptide substrates that have a 4-methoxy-2-naphthylamide (NA) reportergroup attached to a Lysine or Arginine group on a short peptide, andthat hydrolysis could occur across a wide pH range, Experiment 2demonstrates that trichomonal hydrolases can cleave peptide substratesthat have a 7-amido-4-methylcoumarin (AMC) reporter group attached to aLysine or Arginine group on a short peptide.

Experiment 3

This experiment demonstrates that most of the interfering hydrolyticactivity present in vaginal fluid collected from normal, uninfectedwomen is present in the particulate matter and can be largely removed bycentrifugation.

Materials

-   -   Swabs (two per donor) containing vaginal fluid specimens        collected from normal, uninfected women at intervals of 1-2 days        apart    -   Substrate solutions consisting of 8 mM CBZ-Arg-Arg-MNA acetate        (ZRR-MNA), 0.8 mM L-cysteine, and either 100 mM sodium acetate,        pH 5, or 100 mM Tris(hydroxymethyl)aminomethane (TRIS), pH 8    -   Indicator solution consisting of 0.4 mM para-dimethylamino        cinnamaldehyde, 1.4% w/v sodium dodecyl sulfate, and 100 mM        citric acid (pH 4)        Procedure

Pairs of vaginal fluid specimens were obtained from donors on threeseparate days—Day 1, Day 3, and Day 4—and tested on the day ofcollection. The vaginal fluid was extracted from each swab bycentrifugation. The vaginal fluid supernatant from the first swab fromeach donor was used without resuspending the particulate matter that hadformed a pellet in the bottom of the microfuge tubes duringcentrifugation. The vaginal fluid from the second swab from each donorwas mixed vigorously to resuspend the particulate matter to form “whole”vaginal fluid. For each donor, 2 μL of vaginal fluid supernatant wasmixed with 2 μL of pH 5 substrate solution, and 2 μL of vaginal fluidsupernatant was mixed with 2 μL of pH 8 substrate solution on a sheet ofparafilm. Likewise, for each donor, 2 μL of whole vaginal fluid wasmixed with 2 μL of pH 5 substrate solution, and 2 μL of whole vaginalfluid was mixed with 2 μL of pH 8 substrate solution. The sheet ofparafilm was placed into a humidified box to retard evaporation of theliquid drops. After fifteen minutes, 2 μL of indicator solution wasadded to each drop. Ten minutes later, the color of each drop wasvisually scored in 0.5 unit increments using the 0-6 scale presented inTable 2.1 above.

Results

The results for pH 5 and pH 8 are shown in Tables 3.1 and 3.2,respectively. TABLE 3.1 Hydrolase Activity at pH 5 by Color Scale SampleDay 1 Day 3 Day 4 Donor A - whole vaginal fluid 0 0 1 Donor A - vaginalfluid supernatant 0 0 0 Donor B - whole vaginal fluid 0 3 0 Donor B -vaginal fluid supernatant 0 0 0 Donor C - whole vaginal fluid 0 3 1Donor C - vaginal fluid supernatant 0 0 0 Donor D - whole vaginal fluid0 0.5 4 Donor D - vaginal fluid supernatant 0 0.5 1

TABLE 3.2 Hydrolase Activity at pH 8 by Color Scale Day 1 Day 3 Day 4Donor A - whole vaginal fluid 0 1 3 Donor A - vaginal fluid supernatant0 0 0 Donor B - whole vaginal fluid 2.5 3 0 Donor B - vaginal fluidsupernatant 0 0 0 Donor C - whole vaginal fluid 0 4 4 Donor C - vaginalfluid supernatant 0 1 4 Donor D - whole vaginal fluid 0 5 5 Donor D -vaginal fluid supernatant 0 0 0

The activity of interfering hydrolases present in the vaginal fluid ofnormal, uninfected women can vary considerably in unpredictable fashionfrom day to day. For example, at pH 5 hydrolytic activity in the wholevaginal fluid from Donor B was undetectable on Day 1, fairly high twodays later (producing a score of 3), and then undetectable on thefollowing day (Table 3.1). In general, the activity of hydrolasespresent in vaginal fluid was higher at pH 8 than pH 5 (compare Tables3.1 and 3.2). In most cases, the hydrolytic activity was associated withthe particulate matter in the vaginal fluid, and either absent orgreatly decreased in the supernatant portion of the vaginal fluid. Forexample, the hydrolytic activity in whole vaginal fluid from Donor D,when measured at pH 8 on Days 3 and 4, was very high, yet undetectablein the supernatant (Table 3.2). In rare cases, hydrolytic activity washigh in the supernatant also, such as with Donor C on Day 4 (Table 3.2).Some of the vaginal fluid particulate matter from this donor may haveaccidentally been resuspended during processing and therefore present inthe supernatant.

Interpretation

These results show that much of the interfering hydrolytic activitypresent in the vaginal fluid of normal, uninfected women is associatedwith particulate matter in the vaginal fluid such as bacteria, vaginalcells, or other cellular debris. Removal of the particulate matter bycentrifugation or other means greatly decreases the presence ofnon-trichomonal hydrolases in vaginal fluid specimens, thereby providingthe assay with selectivity toward trichomonal hydrolase activity.

Experiment 4

This experiment offers further evidence that most of the interferinghydrolytic activity present in vaginal fluid collected from normal,uninfected women is present in the particulate matter. In thisexperiment, the particulate hydrolases are removed by filtration,specifically centrifugal filtration.

Materials

-   -   Swabs (two per donor) containing vaginal fluid specimens        collected from normal, uninfected women    -   Trichomonal hydrolases prepared as described in Preparation C        above    -   Diluent solution consisting of 50 mM        Tris(hydroxymethyl)aminomethane (TRIS) buffer, pH 8.3, in a 20        mL dropper bottle    -   Polystyrene 1.5 mL cuvettes    -   Nanosep MF centrifugal filtration devices with 0.2 micron pores        (Pall Filtron), each fitted with a disk of coarse filter paper        inside the upper chamber to serve as a prefilter    -   Substrate solution consisting of 4 mM CBZ-Arg-Arg-MNA acetate        (ZRR-MNA) and 1 mM L-cysteine in a 2 mL dropper bottle    -   Indicator solution consisting of 0.8 mM para-dimethylamino        cinnamaldehyde and 2% w/v sodium dodecyl sulfate in a 2 mL        dropper bottle    -   400 mM malic acid, pH 3.4, in a 2 mL dropper bottle        Procedure

To extract the vaginal fluid from each Dacron swab, ten drops(approximately 400 μL) of buffer were placed in a cuvette, then the swabwas inserted into the cuvette and rotated several times. As the swab wasrotated, the narrow portion of the tapered cuvette squeezed the swabhead to thoroughly mix the contents of the swab into the diluentsolution. The swab was removed and discarded. Both swabs from each donorwere treated identically up to this point, then 30 μL of trichomonalhydrolases was added to one of the cuvettes from each pair. Usingdisposable droppers, one drop of unfiltered fluid was transferred fromeach cuvette to separate microwells in a microtiter plate. The remainingfluid in each cuvette was poured into separate centrifugal filterdevices. The filter devices were placed in a microfuge and centrifugedfor five minutes. Using disposable droppers, one drop of filtrate wastransferred from each filter device to separate microwells in themicrotiter plate. One drop of substrate solution was added to eachmicrowell, and the microtiter plate was gently tapped a few times to mixthe contents of the wells. After ten minutes, one drop of indicatorsolution and one drop of malic acid was added to each well, and themicrotiter plate was again gently tapped a few times to mix the contentsof the wells. Five minutes later, the color in each well was visuallyscored in 0.5 unit increments using the 0-6 scale presented in Table 2.1above.

Results

In this experiment, vaginal fluid from only two of the nine donors(Donors F and G) tested positive for hydrolase activity withoutsupplementation with trichomonal hydrolases. Table 4.1 below shows thatfiltration completely removed the hydrolytic activity from these samples(compare the second and third columns of the table). When the vaginalfluid was supplemented with trichomonal hydrolases, all of the vaginalfluid specimens had high levels of hydrolase activity, with littledifference between the unfiltered and filtered solutions (fourth andfifth columns of the table). TABLE 4.1 Hydrolase Activity With andWithout Filtration Vaginal Fluid Vaginal Fluid From Supplemented WithUninfected Subjects Trichomonal Hydrolases Specimens Unfiltered FilteredUnfiltered Filtered Donor A 0 0 4 4 Donor B 0 0 4 4 Donor C 0 0 4 4Donor D 0 0 5 4 Donor E 0 0 4 4 Donor F 3.5 0 5 4 Donor G 0 0 4 4.5Donor H 3.5 0 4 3.5 Donor I 0 0 5 5Interpretation

There were fewer vaginal fluid specimens with detectable hydrolaseactivity in this Experiment than in Experiment 3, probably because ofgreater dilution of the vaginal fluid in this experiment. Theinterfering vaginal fluid hydrolases present in the two specimens withhydrolytic activity were decreased to undetectable levels by filtration,indicating that they are largely insoluble in water. When trichomonalhydrolases were added to the extracted vaginal fluid specimens, thetrichomonal hydrolases passed through the filters readily, resulting inlittle difference between the filtered and unfiltered solutions,suggesting that these trichomonal hydrolases are soluble in water. Asfor many experiments described, the trichomonal hydrolases were added tovaginal fluid rather than tested alone, to insure that only hydrolasesthat are active in the presence of vaginal fluid were being detected.

Experiment 5

This experiment offers still further evidence that most of theinterfering hydrolytic activity present in vaginal fluid collected fromnormal, uninfected women is present in the particulate matter. In thisexperiment, the particulate matter is removed by filtration which isachieved causing the specimens to flow longitudinally through a strip ofporous material.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation B        above    -   Porous material: SMW50 fiber, Manniweb Filtration Systems        Products    -   Plastic drinking straw, cut 2 inches long and heat-sealed shut        at one end    -   Buffer consisting of 100 mM imidazole solution, pH 7.3    -   Dacron swabs containing vaginal fluid specimens collected from a        normal, uninfected woman    -   Substrate solution consisting of 4 mM CBZ-Arg-Arg-MNA acetate        (ZRR-MNA) in 100 mM imidazole, pH 7.3    -   Color developer solution made of a fresh mixture of equal        volumes of the following indicator solution and acid solution:        -   a. Indicator solution: 160 μL of 25 mM para-dimethylamino            cinnamaldehyde in ethanol, 1 mL of 10% w/v sodium dodecyl            sulfate, and 8.8 mL water        -   b. 1M malic acid, pH 3            Procedure

Three strips, 3 mm wide and 45-50 mm long, were cut from the porousmaterial. Each strip was placed on a sheet of parafilm, and two 3mm-wide bands of tape were affixed transversely across each strip. Thefirst band of tape was placed 5 mm from the end of the strip and thesecond band of tape was placed 5 mm from the first band, dividing eachstrip into three sections, i.e., an upstream section at the starting end5 mm in length, a middle section 5 mm in length, and a downstreamsection approximately 30-35 mm in length. Vaginal fluid specimens fromthe same donor were processed three different ways:

1. Extracted whole vaginal fluid: To extract the vaginal fluid from aDacron swab head, 250 μL of buffer was pipetted into a heat-sealed strawand the swab was inserted into the straw. The swab was rotated insidethe straw while pressure was applied to the sides of the straw tothoroughly mix the swab contents into the buffer. The swab was removedfrom the straw slowly while the sides of the straw were squeezed toexpress most of the liquid from the swab head.

Twenty-five μL of the extracted vaginal fluid was slowly pipetted onto a45 mm long porous strip, in the middle section between the two strips oftape. After the extracted vaginal fluid had been completely absorbedinto the strip, 50 μL of buffer was applied dropwise onto the upstreamsection of the strip (at the starting end), allowing the buffer to beabsorbed into the strip between drops. After all of the liquid had beenabsorbed into the material and the entire strip was visibly wetted, thedownstream section of the strip was cut transversely into small slices3-5 mm long. Each slice, in sequence, was transferred to separatemicrowells in a 96-well microtiter plate, and then covered with 25 μL ofsubstrate solution. After ten minutes, 25 μL of color developer solutionwas pipetted into each microwell. Five minutes later, the color thatdeveloped in each well was visually scored in 0.5 unit increments usingthe 0-6 scale presented in Table 2.1 above.

2. Blotted whole vaginal fluid: Instead of extracting the vaginal fluidusing a straw, a swab containing a vaginal fluid specimen from the samedonor was pressed several times onto the middle section of another 45 mmlong porous strip. The swab head was blotted onto the strip until thematerial was flattened and visibly wetted with vaginal fluid. Fifty μLof buffer was pipetted dropwise onto the upstream section of the stripand the strip was processed as above.

3. Blotted whole vaginal fluid plus trichomonal hydrolases: Todemonstrate mobility of trichomonal hydrolases through a porous strip, 4μL of trichomonal hydrolases were pipetted onto the middle section of a50 mm long strip (a longer strip was used to accommodate the additional4 μl of fluid) after a swab as described in Part 2 was blotted onto thesame section. Fifty μL of buffer was pipetted dropwise onto the upstreamsection of the strip and the strip was processed as above.

Results

The results are shown in Table 5.1 below where Section A is the upstreamsection, Section B the middle section, and Sections C2 through C8 theeight subsections of the longer downstream section. TABLE 5.1 HydrolaseActivity Using Filter Strip Strip Section: A B C1 C2 C3 C4 C5 C6 C7 C8Extracted Vaginal Fluid 0 3 0 0 0 0 0.5 0.5 — — Blotted Vaginal Fluid 03 0 0 0 0 0.5 0.5 — — Blotted Vaginal Fluid 0 2.5 0 0 0 0 0.5 2 3 4 PlusTrichomonal Hydrolases

These results show that for the extracted vaginal fluid specimens, asexpected, no hydrolytic activity was detected in the upstream sectionwhere the buffer was applied to the porous strip. Considerablehydrolytic activity was detected in the middle section, which was wherethe vaginal fluid was applied. No activity was detected in the firstfour slices cut from the downstream section of the strip closest to themiddle section (subsections C1-C4), and low levels of hydrolyticactivity were detected in the remaining two slices cut from thedownstream section. The results for the blotted vaginal fluid wereidentical to those for the extracted vaginal fluid. The results fromvaginal fluid supplemented with trichomonal hydrolases were similar forSections A and B and the first four subsections of Section C. Unlike theother strips, however, there was considerable hydrolytic activity in theslices cut from the distal end of Section C (subsections C6-C8), notingonce again that this strip contained two more subsections because of itsgreater length.

Interpretation

Most of the hydrolytic activity present in whole normal vaginal fluidwas detected on the porous strip where the vaginal fluid was applied, inSection B. The lateral flow of buffer from the section at the startingend through the middle section to the downstream section did not causemuch of the hydrolytic activity to flow with the buffer. This is strongevidence that most of the vaginal fluid hydrolases being detected bythis assay are bound to or otherwise associated with particulate matterthat is trapped in or on the porous material. A very small portion ofthe vaginal fluid hydrolases are apparently in a soluble form. Thosethat were soluble were carried along by the flow of buffer to the end ofthe strip. The two methods of transferring the vaginal fluid from theswab to the paper strip had no effect on the amount of hydrolasestrapped by the strip.

When trichomonal hydrolases were added to the vaginal fluid applied to astrip, there was no increase in activity detected in the middle sectionwhere these supplemented specimens were added, but there was a largeincrease in hydrolase activity at the very end of the strip. Thisindicates that trichomonal hydrolases are soluble and can readilymigrate laterally through a porous strip by a flow of liquid in thestrip. This observation indicates that test devices containing a porousstrip to which a vaginal fluid specimen is transferred and which is thenwashed laterally with a buffer are selective for trichomonal hydrolases.The trichomonal hydrolases, but little of the vaginal fluid(particulate) hydrolases, will reach the end of the strip. By placingthe substrate at the end of the strip, hydrolysis of the substrate wouldoccur only if trichomonal (soluble) hydrolases are present in thespecimen.

Experiment 6

This experiment demonstrates that certain protease inhibitors providefurther selectivity to an assay for trichomonal hydrolase activity. Theinhibitors used in this experiment were antipain (an inhibitor of thiolproteases) and chymostatin (an inhibitor of chymotrypsin, a serineprotease). Although these inhibitors can provide a certain degree ofselectivity to assays performed on whole vaginal fluid specimens, theyare particularly useful on vaginal fluid specimens from which theparticulate hydrolases have been removed by filtration orcentrifugation. A relatively low concentration of inhibitor issufficient to eliminate the small amounts of soluble hydrolases that arevariably present in some normal vaginal fluid specimens, and at theconcentrations employed in this experiment, the inhibitors did notsubstantially inhibit the activity of the trichomonal hydrolases.

Materials

-   -   Swabs (two per donor) containing vaginal fluid specimens        collected from normal, uninfected women    -   Trichomonal hydrolases prepared as described in Preparation B    -   Diluent consisting of 10 mM L-cysteine in water    -   0.5 mL centrifuge filter units with 0.2 micron-pore filter        membranes    -   60-well microwell mini tray with 20 μL conical microwells        (originally designed for serotyping)    -   100 mM CBZ-Arg-Arg-MNA acetate (ZRR-MNA) in ethanol    -   1M N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES),        pH 7.6    -   200 μg/mL antipain in water    -   10 mM chymostatin, prepared by adding deionized water to a 330        mM stock made in DMSO    -   Color developer solution made of a fresh mixture of equal        volumes of the following solutions:        -   a. Indicator solution: 320 μL of 25 mM para-dimethylamino            cinnamaldehyde in ethanol, 1 mL of 10% w/v sodium dodecyl            sulfate, and 7.7 mL water        -   b. 400 mM malic acid, pH 3.4            Procedure

1. Demonstration of Strong Inhibition of Non-Trichomonal HydrolasesPresent in Normal Vaginal Fluid Supernatants:

Substrate/inhibitor combinations were prepared by mixing 4 μL of 100 mMZRR-MNA, 20 μL of 1M HEPES buffer, 56 μL of deionized water, and 20 μLof either antipain, chymostatin, or deionized water. Filtered vaginalfluid was prepared from each specimen as follows: the swab was placed ina cuvette containing 200 μL of diluent and the swab was rotated in thediluent to thoroughly mix the swab contents into the diluent. The swabwas removed, the swab head was broken off into a microfuge tube, and theliquid remaining in the swab head was forced out by centrifugation. Thefluid extracted from the swab was combined with the liquid in thecuvette, and the total volume of fluid was filtered using a centrifugefilter unit to remove particulate matter.

For each specimen, 4 μL of vaginal fluid filtrate was mixed with 4 μL ofeach of the three substrate/inhibitor combinations in separatemicrowells; one well contained 20 μg/mL antipain, one contained 1 mMchymostatin, and one had no inhibitor. After ten minutes, 4 μL of colordeveloper solution was added to each well. Ten minutes later, the colorin each well was visually scored in 0.5 unit increments using the 0-6scale presented in Table 2.1 above.

2. Demonstration of Weak Inhibition of Trichomonal Hydrolases:

Trichomonal hydrolases were added to each of the filtered vaginal fluidspecimens—10 μL of trichomonal hydrolase preparation added to 90 μLvaginal fluid filtrate—and the above experiment was repeated.

Results

The results for the vaginal fluid supernatants from normal, uninfectedwomen are shown in Table 6.1 and the results for the vaginalsupernatants that had been supplemented with trichomonal hydrolases areshown in Table 6.2: TABLE 6.1 Hydrolase Activity for Specimens fromUninfected Subjects With and Without Inhibitors Inhibitor: None AntipainChymostatin Donor A 2 0 0 Donor B 0 0 0 Donor C 2 0 0 Donor D 4 0 2Donor E 2 0 0 Donor F 2 0 0

TABLE 6.2 Hydrolase Activity for Specimens Supplemented with TrichomonalHydrolases With and Without Inhibitors Inhibitor: None AntipainChymostatin Donor A 4 4 3.5 Donor B 4 3.5 3.5 Donor C 4 3.5 3.5 Donor D4.5 4 4 Donor E 3 3 3 Donor F 3 2.5 3

1. Demonstration of Strong Inhibition of Hydrolases Present in VaginalFluid Supernatants (Table 6.1)

The results in Table 6.1 show that five of the six normal vaginal fluidspecimens contained hydrolases capable of hydrolyzing ZRR-MNA (secondcolumn of the table). Antipain completely inhibited the hydrolases inall five of these specimens (third column). Chymostatin completelyinhibited the hydrolases in four of the five specimens, and partiallyinhibited the specimen with the highest enzyme activity (fourth column).

2. Demonstration of Weak Inhibition of Trichomonal Hydrolases (Table6.2)

The results in Table 6.2 show that specimens demonstrated increasedhydrolytic activity when augmented with trichomonal hydrolases (secondcolumn). Neither antipain nor chymostatin decreased the intensityproduced by the trichomonal hydrolases by more than a half-unit score(third and fourth columns) compared to the wells that did not contain aninhibitor (second column).

Interpretation

This experiment clearly demonstrates that hydrolase inhibitors atappropriate concentrations selectively inhibit interfering hydrolasesnaturally present in normal vaginal fluid specimens while permittingtrichomonal hydrolases to remain active. This offers a furtherimprovement of the specificity of detection of trichomonal hydrolases.The inhibitors used in this experiment were of two distinct classes:antipain is a thiol protease inhibitor and chymostatin is an inhibitorof chymotrypsin, a serine protease. Each one however provided selectiveinhibition of interfering hydrolases.

Experiment 7

This experiment compares the selective inhibition of water soluble,non-trichomonal hydrolases by two thiol protease inhibitors, E64 andantipain, in vaginal fluid specimens collected from women attending anSTD (sexually transmitted disease) clinic.

Materials

-   -   Swabs (two per donor) containing vaginal fluid specimens        collected from women attending an STD clinic, many of whom had        bacterial vaginosis, vaginal yeast infections, or other forms of        vaginitis, and who were determined to be trichomonas-positive        (vaginally infected with T. vaginalis) or trichomonas-negative        by five-day culture for T. vaginalis    -   Plastic drinking straws, cut to 2-inch lengths and heat-sealed        closed at one end    -   0.5 mL centrifuge filter units with 0.2 micron-pore filter        membranes    -   96-well microwell plates with 250 μL round-bottom wells    -   Substrate reagent solutions containing 4 mM CBZ-Arg-Arg-MNA        acetate (ZRR-MNA), 400 mM imidazole buffer, pH 7.3, and one of        the following two inhibitors:        -   0.4 mM trans-epoxysuccinyl-L-leucylamido-(4-guanidino)            butane (E64)        -   5 μg/mL antipain    -   Color developer solution made of a fresh mixture of equal        volumes of the following solutions:        -   a. Indicator solution: 160 μL of 25 mM para-dimethylamino            cinnamaldehyde in ethanol, 2 mL of 10% w/v sodium dodecyl            sulfate, and 7.8 mL water        -   b. 400 mM malic acid, pH 3.4            Procedure

Each vaginal fluid specimen from a clinic patient was placed inside aheat-sealed plastic straw containing 250 μL of deionized water. The swabwas rotated while the straw was being pinched, to thoroughly mix theswab contents into the water, then the swab was removed while the strawwas being squeezed to express most of the liquid from the swab tip. Toremove particulate matter, the extracted vaginal fluid was transferredto a filter unit and centrifuged for up to five minutes in a smallportable centrifuge. Twenty-five μL of the substrate solution containingE64 was pipeted into a round-bottom microwell, and 25 μL of thesubstrate solution containing antipain was pipeted into a second well.Twenty-five μL of the filtered vaginal fluid was added to each of twowells. After ten minutes, 25 μL of color developer solution was added toeach well. Five minutes later, the color in each well was visuallyscored in 0.5 unit increments using the 0-6 scale presented in Table2.1.

Results

The results are shown in Table 7.1: TABLE 7.1 Hydrolase Activity inSpecimens from Trichomonas-Positive and Trichomonas-Negative SubjectsWith Inhibitors Inhibitor: Color Score E64 Antipain Trichomonas-positive(n = 5): 0 2 2 1-1.5 0 0 2-2.5 1 1 3-3.5 0 0 4-4.5 2 2 5-6   0 0Trichomonas-negative (n = 21): 0 21 17 1-1.5 0 2 2-2.5 0 2 3-3.5 0 04-4.5 0 0 5-6   0 0

Three of five specimens that were culture-positive for trichomonasproduced a pink color in the test wells (upper half of Table 7.1). Forthese specimens, the choice of inhibitor did not affect the colorscores; i.e., the wells containing E64 scored nearly the same (within ahalf-unit) as the matching wells containing antipain. The twoculture-positive specimens that failed to produce a pink color had lownumbers of parasites: only a few trichomonads were seen in themicroscopic wet mount examination from one, and none were seen in thewet mount from the other specimen. All twenty-one culture-negativespecimens scored zero in the wells containing E64, and eighteen of thetwenty-one culture-negative specimens scored zero in the wellscontaining antipain (lower half of Table 7.1).

Interpretation

This experiment was designed to compare the efficacy of two inhibitors.Only two specimens were available from each donor at this clinic, so itwas not possible to compare the two inhibitors while also testing thehydrolase activity in the absence of an inhibitor. As demonstrated inExperiment 6, however, a certain amount of non-trichomonal hydrolaseactivity may remain in vaginal fluid specimens after filtration, andantipain can be used to selectively inhibit this non-trichomonalhydrolase activity. Experiment 7 establishes that a different thiolhydrolase inhibitor, E64, also can be very effective at selectivelyinhibiting non-trichomonal hydrolases that may be present in vaginalfluid specimens. At the concentrations of inhibitor tested, E64 was moreeffective that antipain at selectively inhibiting non-trichomonalhydrolases present in vaginal specimens from clinic patients withnon-trichomonal vaginal infections, while permitting trichomonalhydrolases to remain active.

Experiment 8

This experiment shows that certain polypeptides can be used toselectively inhibit non-trichomonal hydrolases present in vaginal fluid,with relatively little inhibition of trichomonal hydrolases while otherpolypeptides do not have these effects.

Materials

-   -   Swabs containing vaginal fluid specimens collected from four        normal, uninfected women    -   Trichomonal hydrolases prepared as described in Preparation A    -   60-Well microwell mini-tray with 20 μL conical microwells    -   Substrate/polypeptide solutions containing 8 mM CBZ-Arg-Arg-MNA        acetate (ZRR-MNA), 0.8 mM L-cysteine, 56 mM acetate (pH 5) or        TRIS (pH 8) buffer, and 10 mM of one of the following        polypeptides:        -   Lys-Arg-Gln-His-Pro-Gly (KRQHPG)        -   Lys-Pro-Gln-Leu-Trp-Pro (KPQLWP)        -   Arg-Lys-Asn-Val-Tyr (RKNVY)        -   Arg-Pro-Lys-Pro-Gln-Phe-Phe-Gly-Leu-Met (RPKPQFFGLM)        -   No polypeptide (control)    -   Indicator solution consisting of 0.4 mM para-dimethylamino        cinnamaldehyde, 1.4% w/v sodium dodecyl sulfate, and 100 mM        citric acid (pH 4)        Procedure

The vaginal fluid was extracted from each swab by centrifugation, andthen mixed to resuspend the particulate matter. For each donor, 2 μL ofvaginal fluid was pipetted into each of ten microwells, then 2 μL of oneof the ten substrate/buffer/polypeptide combinations was added to eachwell. Additionally, 2 μL of trichomonal hydrolases was mixed with 2 μLof each of the substrate/polypeptide combinations in set of ten wells.After fifteen minutes, 2 μL of indicator solution was added to eachwell. Ten minutes later, the color in each well was visually scored in0.5 unit increments using the 0-6 scale presented in Table 2.1.

Results

The results for the tests at pH 5 are shown in Table 8.1 and those forthe tests at pH 8 are shown in Table 8.2: TABLE 8.1 Hydrolase Activityin Specimens from Uninfected Women Using Different Peptide Inhibitors atpH 5 Donor Donor Donor Trichomonal A B Donor C D Hydrolases Nopolypeptide 3 3 0 3 3 KPQLWP 0 0 0 0 3 RKNVY 0.5 0 0 0 3 KRQHPG 2 2 0 23 RPKPQFFGLM 0.5 1 0 0.5 1

TABLE 8.2 Hydrolase Activity in Specimens from Uninfected Women UsingDifferent Peptide Inhibitors at pH 8 Donor Donor Donor Trichomonal A BDonor C D Hydrolases No polypeptide 3 3 2.5 3 3 KPQLWP 0.5 0.5 0 0 3RKNVY 0.5 0.5 1 1 3 KRQHPG 2.5 2 0.5 2 3 RPKPQFFGLM 0.5 0.5 0 0 0.5

These tables show that when the assays were run at pH 5, the vaginalfluid from three of the four donors contained sufficient hydrolyticactivity to produce color scores of 3 (the “No polypeptide” row in Table8.1). The trichomonal hydrolases also produced a score of 3 in thisassay. In presence of either KPQLWP or RKNVY, the vaginal fluidhydrolases produced very little color, with all but one well scoringzero, while the trichomonal hydrolases remained unaffected. When theassays were run at pH 8, the vaginal fluid from all four donors, as wellas the trichomonal hydrolases, each produced color scores of 2.5 or 3.Again, KPQLWP and RKNVY inhibited color production by vaginal fluidhydrolases at this pH, but not as much as at pH 5. The polypeptide,KRQHPG, had weak inhibitory effects on the color production by vaginalfluid hydrolases at either pH tested. In contrast, the largestpolypeptide, RPKPQFFGLM, inhibited the color produced by both thevaginal fluid hydrolases and the trichomonal hydrolases, with no wellsscoring greater than 1.

Interpretation

The polypeptide RPKPQFFGLM did not exhibit the desired property ofselectively inhibiting non-trichomonal vaginal fluid hydrolases, as itdecreased the hydrolysis of the substrate (ZRR-MNA) by both trichomonalhydrolases and normal vaginal fluid hydrolases. The other three peptidesexamined did not decrease color production by trichomonal hydrolases.One of these, KRQHPG, had little inhibitory effect on the vaginal fluidhydrolases but the remaining two polypeptides, KPQLWP and RKNVY,demonstrated selective inhibition of vaginal fluid hydrolases,especially at pH 5.

Experiment 9

This experiment compares certain dipeptides in their ability toselectively inhibit non-trichomonal hydrolases present in whole vaginalfluid and vaginal fluid supernatant with relatively little inhibition oftrichomonal hydrolases.

Materials

-   -   Swabs containing vaginal fluid specimens collected from four        normal, uninfected women    -   Trichomonal hydrolases prepared as described in Preparation A    -   60-well microwell mini-tray with 20-μL conical microwells    -   Substrate/polypeptide solutions containing 8 mM CBZ-Arg-Arg-MNA        acetate (ZRR-MNA), 0.8 mM L-cysteine, 56 mM acetate (pH 5) or        TRIS (pH 8) buffer, and 10 mM of one of the following        dipeptides:        -   Lys-proline        -   Lys-valine        -   Lys-Lys        -   No dipeptide (control)    -   Indicator solution consisting of 0.4 mM para-dimethylamino        cinnamaldehyde, 1% w/v sodium dodecyl sulfate, and 20 mM malic        acid (pH 3.4)        Procedure

For each specimen, the vaginal fluid was extracted from the swab bycentrifugation, 2 μL of the vaginal fluid supernatant was removed fromthe microfuge tube, and the remainder of the fluid was mixed toresuspend the particulate matter. For each specimen, 2 μL of the thusreconstituted vaginal fluid was pipetted into each of four microwells,then 2 μL of one of the four substrate/dipeptide combinations was addedto each well. The 2 μL of vaginal fluid supernatant from each specimenwas mixed with 18 μL of trichomonal hydrolases, and 2 μL of this mixturewas mixed with 2 μL of each of the substrate/dipeptide combinations inanother set of four wells. After fifteen minutes, 2 μL of indicatorsolution was added to each well. Ten minutes later, the color in eachwell was visually scored in 0.5 unit increments using the 0-6 scalepresented in Table 2.1.

Results

The results for the tests performed on whole vaginal fluid are shown inTable 9.1 and those for the tests performed with vaginal fluidsupernatant are shown in Table 9.2: TABLE 9.1 Hydrolase Activity inWhole Vaginal Fluid Specimens from Uninfected Women Using DifferentDipeptide Inhibitors Donor A Donor B Donor C Donor D No dipeptide 0.5 00.5 2 Lysine-proline 0 0 0 0 Lysine-valine 1 0 0 1.5 Lysine-lysine 1 0 02.5

TABLE 9.2 Hydrolase Activity in Vaginal Fluid Supernatant Specimens fromUninfected Women Using Different Dipeptide Inhibitors - TrichomonalHydrolase Added Donor A Donor B Donor C Donor D No dipeptide 1 2 1 2.5Lysine-proline 1 1 0.5 2 Lysine-valine 1 2 1 2 Lysine-lysine 1 2 1 2

These tables show that three of the four whole vaginal fluid specimenscontained measurable hydrolytic activity (“No dipeptide” row in Table9.1). The dipeptide, lysine-proline, completely inhibited colorproduction by all of those three whole vaginal fluid specimens. Theother two dipeptides tested, lysine-valine and lysine-lysine, had littleeffect on the color produced by the whole vaginal fluid specimens; allof the wells containing either of these dipeptides scored within 0.5 ofthe corresponding control well for each vaginal fluid donor. Mixtures ofnormal vaginal fluid supernatants with trichomonal hydrolases producedcolor scores of one to 2.5 (“No dipeptide” row 1 in Table 9.2). Thepresence of lysine-proline decreased the color scores produced bytrichomonal hydrolases slightly, while lysine-valine or lysine-lysinehad little or no inhibitory effect.

Interpretation

Two of the three dipeptides tested, lysine-valine and lysine-lysine, hadlittle effect on the color produced by either vaginal fluid hydrolasesor trichomonal hydrolases (color score differences of only 0.5 can beattributed to variations in the amount of particulate matter pipetted toeach well). Lysine-proline, however, demonstrated selective inhibitionof vaginal fluid hydrolases, with only a small degree of inhibition oftrichomonal hydrolases.

Experiment 10

This experiment illustrates the use of a test device containing a stripof filter paper impregnated with the indicatorpara-dimethylamino-cinnamaldehyde, a substrate conjugate,CBZ-Arg-Arg-MNA (ZRR-MNA), and the enzyme inhibitor E64 in tests onvaginal fluid specimens collected from women attending an STD clinic.

Materials

-   -   Swabs (two per donor) containing vaginal fluid specimens        collected from women attending an STD clinic, many of whom had        bacterial vaginosis, vaginal yeast infections, or other forms of        vaginitis, and which were determined to be trichomonas-positive        (vaginally infected with T. vaginalis) or trichomonas-negative        by five-day culture for T. vaginalis    -   100 mM imidazole buffer, pH 7.1    -   40 mM CBZ-Arg-Arg-MNA (ZRR-MNA) and 0.2 mM        trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64) in        ethanol    -   24 mM para-dimethylamino-cinnamaldehyde (pDMAC) and 5% (w/w)        hydroxy-propylcellulose (HPC) in ethanol    -   3M maleic acid, 2% (w/v) sodium dodecyl sulfate, and 6% (w/v)        hydroxypropylcellulose in water        Procedure

Referring to FIGS. 3 a through 3 d, test devices were assembled asfollows: Two 1-inch-long strips of double-sided tape were affixed inparallel, ¾-inch apart, to a sheet of Mylar 11 that was 5 mils thick. A3/16-inch×⅝-inch strip of Ahlstrom 237 filter paper 12 was affixed tothe Mylar sheet with one end (the “upper” end, or the right end as shownin the Figures) of the filter paper strip aligned with the ends of thestrips of tape. Approximately 1 μL of the pDMAC mixture was applied as acoating 13 to the upper half of the filter paper strip and allowed todry. Two μL of the ZRR-MNA mixture 14 was pipetted evenly over ¼-inch ofthe opposite end of the filter paper strip and allowed to dry. A secondsheet of Mylar 20 was affixed to the first sheet, held together by thetwo strips of double-sided tape, forming a flat sleeve. The filter paperstrip 12 was located between the Mylar sheets along the portions thatformed the sleeve. A third sheet of Mylar 16, previously coated with athin dried film 17 of the maleic acid mixture, was affixed to the firstsheet of Mylar 11 such that the coated surface was overlying the filterpaper strip 12, but temporarily kept from contact with the paper by thesecond sheet of Mylar 20.

Each swab containing a vaginal fluid specimen from a clinic patient wasplaced into the sleeve section of a test device. While the test devicewas held horizontally, a pipet was inserted into the sleeve and 140 μLof buffer was carefully pipetted onto the swab head. The swab wasrotated ten times to mix the buffer into the swab head evenly. The swabwas inserted deeper into the sleeve until the swab head made firmcontact with the end of the filter paper strip, allowing fluid from theswab head to be wicked slowly into the filter paper. After fifteenminutes, the end of the test device was bent and then permitted to snapback, which brought the acid film in contact with the filter paperstrip. The reagents in the acid film facilitate a indicator reactionbetween para-dimethylamino-cinnamaldehyde and any product (MNA) producedby hydrolysis of the substrate by hydrolases present in the specimen.After a five-minute wait, the test device was examined for thedevelopment of a pink line on the filter paper strip in the region wherethe substrate was located.

Results

The results are shown in Table 10.1. TABLE 10.1 Hydrolase Activity inVaginal Fluid Specimens Subjects Attending an STD Clinic Using TestDevice Number of Color Specimens Trichomonas-positive (n = 3): No pinkcolor 0 Faint pink color 0 Distinct pink color 3 Trichomonas-negative (n= 6): No pink color 5 Faint pink color 1 Distinct pink color 0

Three of the ten vaginal fluid specimens tested were obtained from womenwith trichomoniasis. The table shows that all three produced a positivetest, forming a pink line on the paper strip in the test device. Five ofthe six specimens from women without trichomoniasis failed to produceany pink color, and one of the six produced only a faint pink color.

Interpretation

This experiment demonstrates that a simple device containing a filterpaper strip impregnated with a substrate, indicator and inhibitor iscapable of accurately detecting trichomonal hydrolases in vaginal fluidspecimens. Other than the buffer solution, all of the reagents were indry form inside the device. All steps were performed within the device,including dilution of the vaginal fluid specimen with buffer, filtrationof the vaginal fluid, hydrolysis of the substrate, and reaction withindicator to produce a visible color. The vaginal fluid, mixed withbuffer, was drawn into the filter paper from the swab by wicking. As thefluid traveled laterally through the porous support, the particulatematter was filtered out, and the indicator was mixed in. After thismixture reached the zone containing the substrate and inhibitor,hydrolysis of the substrate occurred if trichomonal hydrolases werepresent in the vaginal fluid. A fifteen-minute wait provided enough timefor the fluid to flow down the strip and for a sufficient amount ofsubstrate to be hydrolyzed by a trichomonas-positive specimen. Afteracid was applied to the strip, the product of hydrolysis of thesubstrate reacted with the indicator to produce a clearly visible darkpink line on the strip to indicate a positive result.

Experiment 11

This experiment compares the hydrolysis of CBZ-Arg-Arg-Arg-MNA(ZRRR-MNA) by trichomonal hydrolases at various pH levels over a pHrange of 1.9 to 3.4. This experiment also demonstrates a procedure inwhich a swab is applied to a dried substrate film, then ten minuteslater to an indicator film, and the result read directly on the swab.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D,        then diluted to 10% v/v by mixing 50 μL trichomonal hydrolases        with 5 μL of 100 mM L-cysteine hydrochloride and 445 μL water    -   100 mM DL-malic acid solutions adjusted to pH 1.3, 1.6, 1.9,        2.2, 2.5, 2.8, 3.1 or 3.4 using HCl    -   200 mM CBZ-Arg-Arg-Arg-MNA triacetate (ZRRR-MNA) in ethanol    -   25% (w/w) hydroxypropylcellulose in ethanol    -   Indicator films prepared as described in Preparation G using 100        mM Fast Red RL dye        Procedure

The 200 mM ZRRR-MNA solution was mixed with 25% hydroxypropylcellulosesolution and additional ethanol to a final concentration of 80 mMZRRR-MNA and 5% hydroxypropyl-cellulose. Substrate films were made bypipetting 1 μL of a mixture over a circular area approximately ¼-inch indiameter on a sheet of Mylar, and drying under a stream of dry nitrogen.An 80-μL aliquot of a malic acid buffer solution, at each pH listedabove, was pipetted into each of eight separate round-bottom wells on amicrotiter plate. Eighty μL of 10% trichomonal hydrolase solution wasadded to each well and mixed thoroughly. Half of the volume in each well(80 μL) was then transferred to a second set of eight wells, into whichthe swabs were then inserted. After the 80 μL of liquid was absorbedfrom each well into the respective swab, each swab was rubbed on asubstrate film. Ten minutes after the swabs were exposed to substrate,each swab was rubbed on an indicator (Fast Red) film. The intensity ofpink color that developed on each swab after one minute was visuallyscored in 0.5 unit increments using the 0-9 color scale presented inTable 11.1 below. To determine the final pH of the eight mixtures oftrichomonal hydrolases and buffer, the pH of the liquid remaining in thefirst set of eight wells was measured using a pH meter. The experimentwas repeated omitting the lowest pH and highest pH wells. TABLE 11.1Fast Red RL Color Scores Color Score Colorless or yellow - no pink color0 Peach or orange trace Faint pink 1 Intermediate intensities of pink2-8 Very intense pink/red 8Results

The results are shown in Table 11.2, and they indicate that the highesthydrolytic activity occurred at approximately pH 2.4: TABLE 11.2Trichomonal Hydrolase Activity at Varying pH pH of 100 mM malicacid/final pH in wells Color Scores: 1.3/1.9 1.6/2.1 1.9/2.3 2.2/2.42.5/2.6 2.8/2.9 3.1/3.2 3.4/3.4 1st Experiment 2 4.5 5 5.5 5.5 5 4.5 3Duplicates — 4.5 5 6 5 3.5 3 —Interpretation

The data in Table 11.2 show that enzymes present in a trichomonalhydrolase solution can hydrolyze ZRRR-MNA at low pH, with a peak ofactivity of approximately pH 2.4. This feature of trichomonal hydrolasescan be exploited in simple diagnostic test that is selective fortrichomoniasis, since most hydrolases function very poorly at this lowpH.

Experiment 12

This experiment compares the hydrolysis of CBZ-Arg-Arg-Arg-MNA(ZRRR-MNA) by trichomonal hydrolases in four different buffers betweenpH 2.0 to 2.6.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D,        then diluted to 10% v/v by mixing 100 μL trichomonal hydrolases        with 10 μL of 100 mM L-cysteine hydrochloride and 890 μL water    -   100 mM L-cysteine hydrochloride    -   100 mM buffer solutions, each at pH 2.0, 2.2, 2.4, and 2.6        -   a. DL-malic acid        -   b. glycline        -   c. L-threonine        -   d. L-lysine    -   8 mM CBZ-ArgArg-Arg-MNA (ZRRR-MNA) prepared by dilution in water        of 200 mM stock in ethanol    -   Indicator films prepared as described in PREPARATION G using 100        mM Fast Red RL dye        Procedure

Fifty μL of each buffer solution, at pH 2.0, 2.2, 2.4, or 2.6, waspipetted into separate wells of a microtiter plate. Twenty-five μL of 8mM ZRRR-MNA and 25 μL of 10% trichomonal hydrolase solution were addedto each well, and 20 μL were then removed from each well for measurementof final pH. Ten minutes after the mixtures were made, a Dacron swab wasinserted into each well to absorb the liquid, then each swab was rubbedon a Fast Red RL film. The intensity of pink color that developed oneach swab after five minutes was visually scored in 0.5 unit incrementsusing the 0-9 color scale presented in Table 11.1 of the precedingexample.

Results

The results are shown in Table 12.1, which indicates that regardless ofthe buffer used, the final pH in each well was 0.3 to 0.5 pH unitshigher than the respective 100 mM buffer stock. Threonine provided thebest overall pH control, with the smallest difference between pH of thebuffer solution and final pH of the mixture in the wells. At any givenfinal pH, the color scores were the highest with threonine as thebuffer: at pH 2.4, the well with threonine scored 6.0 as compared to 5.0for glycine and 4.5 for malic acid; at pH 2.7, the threonine well scored6.5 as compared to 6.0 for glycine and 5.5 for malic acid; at pH 2.9,the wells with threonine, glycine or malic acid all scored 6.0. TABLE12.1 Trichomonal Hydrolase Activity at Varying pH pH 2.0 Buffer pH 2.2Buffer pH 2.4 Buffer pH 2.6 Buffer Final pH Score Final pH Score FinalpH Score Final pH Score DL-malic pH 2.4 4.5 pH 2.7 5.5 pH 2.8 6.0 pH 2.96.0 acid Glycine pH 2.4 5.0 pH 2.6 6.0 pH 2.7 6.0 pH 2.9 6.0 L-threoninepH 2.3 3.5 pH 2.4 6.0 pH 2.7 6.5 pH 2.9 6.0 L-lysine pH 2.3 3.0 pH 2.54.5 pH 2.9 5.0 pH 3.0 5.5Interpretation

Experiment 11 demonstrated maximal enzyme activity by trichomonalhydrolases at pH 2.4. The ideal buffer would support maximal enzymeactivity by trichomonal hydrolases while providing adequate bufferingcapacity at this pH. Insufficient buffering, with the resultant higherpH, could lead to both decreased activity by trichomonal hydrolases andincreased activity by non-trichomonal hydrolases. Furthermore, highconcentrations of buffer may inhibit trichomonal hydrolase activity.

The present experiment shows that all four of the buffers tested at pH2.4 were compatible with trichomonal hydrolase activity, although enzymeactivity was not as high with lysine as with the other three buffers.The best buffering capacity under the conditions of this test wasdemonstrated by threonine and malic acid. Between these two buffers,threonine provided the best pH control and supported the highesthydrolase activity.

Experiment 13

This experiment tests compares a series of peptide substrate conjugatesin terms of the ability of trichomonal hydrolases to hydrolyze thesesubstrates at low pH.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D    -   200 mM L-threonine buffer, pH 2.4    -   2 mM L-cysteine hydrochloride in water    -   Pooled normal vaginal fluid supernatant (NVS) prepared as        follows: vaginal fluid specimens collected on dacron swabs from        normal, uninfected women were centrifuged to extract the        undiluted fluid from the swabs and to pellet the particulate        matter, and then the supernatants were pooled and frozen until        use    -   0.2 mg/mL Fast Garnet GBC sulfate, dissolved in water no more        than an hour before being used    -   2% sodium dodecyl sulfate in 500 mM pH 5 acetate buffer        (SDS/acetate)    -   2 mM solutions of each of a series of substrates listed below in        threonine buffer; hydrophobic peptide substrates were initially        dissolved in a small amount of DMSO, then diluted to 2 mM in        threonine buffer        Procedure

1. Assays Without Vaginal Fluid Present

Forty μL of each substrate was pipetted into duplicate wells of amicrotiter plate. The plate was covered with tape, placed into a 25° C.incubator, and allowed to warm. A diluted enzyme mixture was prepared bymixing one part (v/v) freshly-thawed trichomonal hydrolase solution tonine parts 2 mM L-cysteine solution. Forty μL of this mixture was addedto one well of each pair of duplicate wells. For negative controls, 40μL of 2 mM L-cysteine was added to the second well in each pair ofduplicate wells. The plate was returned to the incubator and incubatedfor ten minutes. Forty μL of SDS/acetate solution was added to eachwell, followed by 40 μL of Fast Garnet solution. The intensity of colorthat developed in each well after five minutes was visually scored usingthe color scale presented in Table 13.1. TABLE 13.1 Color Scores ColorScore Interpretation − No red color produced + Clearly visible red colorproduced ++ Moderately intense red color produced +++ Very intense redcolor producedResults

1. Assays Without Vaginal Fluid Present

Test results for 61 peptide substrates are listed in Table 13.2. Ofthese substrates, only thirteen were hydrolyzed by trichomonalhydrolases. Of the thirteen substrates hydrolyzed, only nine producedsuitably intense color (scoring either ++ or +++). TABLE 13.2Trichomonal Hydrolase Activity on Various Peptide Substrates PeptideSubstrate Color Score D-Val-Leu-Arg-MNA +++ CBZ-Arg-Arg-Arg-MNA +++CBZ-Leu-Arg-MNA +++ CBZ-Val-Arg-MNA +++ CBZ-Phe-Arg-MNA ++BZ-Phe-Val-Arg-MNA ++ CBZ-Arg-Arg-MNA ++ D-Val-Leu-Lys-MNA ++CBZ-Ala-Arg-Arg-MNA ++ CBZ-Val-Leu-Arg-MNA + CBZ-Leu-Arg-Arg-MNA +CBZ-Val-Lys-Lys-Arg-MNA + CBZ-Lys-Lys-Arg-MNA + CBZ-Lys-βNA −BZ-DL-Arg-βNA − BZ-Arg-MNA − CBZ-Arg-MNA − Arg-Arg-βNA − CBZ-Arg-Arg-βNA− BZ-Pro-Phe-Arg-βNA − BOC-Gln-Ala-Arg-MNA − CBZ-Gly-Gly-Arg-MNA −CBZ-Gly-Pro-Arg-MNA − Phe-Arg-βNA − Pro-Arg-MNA − Gly-Arg-βNA −Gly-Arg-MNA − BOC-Gln-Ala-Ala-MNA − CBZ-Arg-Gly-Phe-Leu-MNA −CBZ-Gly-Gly-Leu-βNA − CBZ-Gly-Gly-Phe-βNA − Gln-Gly-Phe-MNA −Ala-Ala-βNA − Gly-Phe-MNA − Gly-Phe-βNA − Gly-Gly-βNA − Gly-Pro-MNA −Gly-Trp-βNA − His-Ser-MNA − Leu-Ala-βNA − Lys-Ala-βNA − Ser-Met-βNA −Ser-Tyr-βNA − Arg-βNA − Arg-MNA − Lys-βNA − Ala-βNA − Gly-βNA − His-βNA− Ile-βNA − Leu-βNA − Leu-MNA − Leu-MNA − Phe-βNA − Phe-MNA − Pro-βNA −Pro-MNA − Hyp-βNA − Trp-βNA − Ser-βNA − Val-βNA −

2. Assays Without Vaginal Fluid Present

Of the nine substrates hydrolyzed by trichomonal hydrolases to producean intense pink color (scoring ++ or +++) in the absence of vaginalfluid supernatant, only four produced intense pink color when vaginalfluid supernatant was present. These results are listed in Table 13.3.TABLE 13.3 Trichomonal Hydrolase Activity on Peptide Substrates inVaginal Fluid Supernatant Peptide Substrate Color ScoreD-Val-Leu-Arg-MNA +++ CBZ-Arg-Arg-Arg-MNA ++ CBZ-Leu-Arg-MNA +++CBZ-Val-Arg-MNA ++ CBZ-Phe-Arg-MNA + BZ-Phe-Val-Arg-MNA +CBZ-Arg-Arg-MNA + D-Val-Leu-Lys-MNA + CBZ-Ala-Arg-Arg-MNA +Interpretation

Only thirteen of the 61 peptide substrates tested generated color in thepresence of trichomonal hydrolases at very low pH. Of these, only nineproduced sufficient color. Four of these nine substrates were readilyhydrolyzed by trichomonal hydrolases in the presence of vaginal fluid:D-Val-Leu-Arg-MNA (VLR-MNA); CBZ-Leu-Arg-MNA (ZLR-MNA);CBZ-Arg-Arg-Arg-MNA (ZRRR-MNA); and CBZ-Val-Arg-MNA (ZVR-MNA).

Experiment 14

This experiment compares vaginal fluid specimens from normal, uninfectedwomen with vaginal fluid specimens to which trichomonal hydrolases havebeen added, in terms of the action of these specimens on four peptidesubstrates at low pH. This example also illustrates a test procedureinvolving applying a swab with specimen to a substrate and indicator andsubsequently reading the result directly on the swab.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D    -   Cotton swabs (two per donor) containing vaginal fluid specimens        collected from normal, uninfected women.    -   200 mM L-threonine buffer, pH 2.4    -   Peptide substrates:        -   400 mM D-Val-Leu-Arg-MNA (VLR-MNA) in ethanol with 10% (v/v)            6M HCl        -   400 mM CBZ-Leu-Arg-MNA (ZLR) in methanol with 10% (v/v)            dimethylformamide        -   400 mM CBZ-Arg-Arg-Arg-MNA (ZRRR-MNA) in ethanol        -   400 mM CBZ-Val-Arg-MNA (ZVR) in methanol with 10% (v/v)            dimethylformamide    -   2 mM L-cysteine hydrochloride in water    -   25% (w/w) hydroxypropylcellulose in ethanol    -   Indicator films prepared as described in PREPARATION G using        either 10 mM Fast Garnet or 70 mM Fast Red RL diazonium dye        Procedure

1. Preparation of Substrate Films

Mixtures were made of the 400 mM substrate stock solutions, 25%hydroxy-propylcellulose solution, 200 mM L-cysteine, along withadditional ethanol to a final concentration of 150-320 mM substrate, 20mM L-cysteine, and 2-5% hydroxypropyl-cellulose. Substrate films weremade by pipetting 1 μL of a mixture over a circular area approximately14-inch in diameter on a sheet of Mylar, and drying under a stream ofdry nitrogen. The dried films were stored in a container with desiccantuntil used.

2. Comparison of Substrates Using Vaginal Fluid Specimens on CottonSwabs

For each pair of vaginal fluid specimens from a donor, one swab wasrubbed on one substrate film and the other swab was rubbed on adifferent substrate film; two substrates were compared at a time. Theconcentrations of the substrates varied from 150 mM to 320 mM in thesolutions used to make the substrate films, but each specific comparisonmatched equimolar concentrations of substrates (as specified in Tables14.1, 14.2, and 14.3 below). In the swabs, the substrate was diluted byvaginal fluid and buffer, and the final concentration of substrate inthe swab tip was estimated to be 1/60 of the concentration of substratein the solutions used to make the films. Immediately after each swab wasrubbed on a substrate film, a 35-μL drop of threonine buffer was appliedto the plastic where the substrate film had been, and the swab wasrubbed on the same spot again until the buffer was absorbed into theswab. The swabs were left lying on the plastic for ten minutes, theneach swab was rubbed on a Fast Red or Fast Garnet film. For thecomparisons between ZLR and ZVR, and between VLR-MNA and ZRRR-MNA, filmsmade with 10 mM Fast Garnet were used; for the comparison between ZVRand VLR-MNA, films made with 70 mM Fast Red were used. The intensity ofpink color that developed on each swab after two minutes was visuallyscored using the 0-9 color scale presented in Table 11.1 above;fractional scores such as 1.3 were used when color intensities betweenswabs differed only slightly.

3. Comparison of Substrates Using Trichomonal Hydrolases Added toVaginal Fluid Specimens on Cotton Swabs

In this part of the experiment, 20 μL of trichomonal hydrolases wasapplied to the tip of each swab containing vaginal fluid just prior torunning the assay. Otherwise, the procedure was exactly the same as forthe swabs containing only vaginal fluid.

Results

The results are shown in the following tables: Table 14.1 compares ZLRwith ZVR at 320 mM, Table 14.2 compares VLR-MNA with ZVR at 150 mM, andTable 14.3 compares VLR-MNA with ZRRR-MNA at 200 mM. TABLE 14.1 PeptideSubstrate Comparisons ZLR ZVR Vaginal Fluid Specimens 320 mM 320 mMNormal (n = 4) 0 to Trace Trace to 1.5 Hydrolases added (n = 4) Trace to1 1 to 5

TABLE 14.2 Peptide Substrate Comparisons VLR-MNA ZVR Vaginal FluidSpecimens 150 mM 150 mM Normal (n = 3) 0 2.5 to 3 Hydrolases added (n =2) 2 to 2.5 4.5 to 5

TABLE 14.3 Peptide Substrate Comparisons VLR-MNA ZRRR-MNA Vaginal FluidSpecimens 200 mM 200 mM Normal (n = 6) 0 to Trace 0 to Trace Hydrolasesadded (n = 5) 1 to 4 1 to 4

Table 14.1 shows that when ZLR was compared with ZVR at 320 mM, thecolor produced by ZLR was too faint in swabs containing vaginal fluidplus trichomonal hydrolases, scoring only one or less. ZVR scored 1 orhigher in all of the specimens with hydrolases added, but also scored ashigh as 1.5 in normal vaginal fluid specimens without hydrolases added.

In Table 14.2, the concentration was reduced from 320 mM to 150 mM in anattempt to decrease the sensitivity of ZVR to endogenous hydrolasespresent in normal vaginal fluid. The results in this table show that ZVRagain produced pink color in normal specimens, this time scoring over 2,while VLR-MNA produced scores of zero in normal specimens, and scores of2 or higher in specimens to which trichomonal hydrolases had been added.

In Table 14.3, VLR-MNA is compared with ZRRR-MNA at an increasedconcentration of 200 mM to improve the color intensity from trichomonalhydrolases. Both substrates produced zero to trace color in swabscontaining only vaginal fluid, and color scores of 1 or more in swabscontaining vaginal fluid with trichomonal hydrolases added.

Interpretation

For clinical utility, it is essential that the substrates produce nomore than trace color (less than a score of 1) in specimens containingvaginal fluid from normal, uninfected women, and color intensitiesscoring at least 1 in swabs to which trichomonal hydrolases have beenadded to the vaginal fluid. ZLR proved to be a poor substrate comparedto ZVR, sometimes producing color intensity scores of less than 1 inswabs which had trichomonal hydrolases added to the vaginal fluid. ZVR,in turn, was less desirable than VLR-MNA, due to unacceptably highreactivity with hydrolases in present in normal vaginal fluid specimens,scoring as high as three in these swabs. Both VLR-MNA and ZRRR-MNAproduced favorable results in this experiment.

Experiment 15

This experiment compared two testing procedures using vaginal fluidspecimens with trichomonal hydrolases added to the swabs: a) applicationof the diazonium dye to the swab immediately after the swab was rubbedon the substrate film (“one-phase” application of reagents to the swab),and b) application of the diazonium dye to the swab ten minutes afterthe swab was rubbed on the substrate film (“two-phase” application ofreagents to the swab). The goal was to determine if the nearlysimultaneous “one-step” application of the substrate and diazonium dyewould affect the intensity of pink color produced.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D    -   Cotton swabs (two per donor) containing vaginal fluid specimens        collected from normal, uninfected women    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation E using 80        mM ZRRR-MNA    -   Indicator films prepared as described in Preparation G using 10        mM Fast Garnet diazonium dye        Procedure

Immediately before each assay, 10 μL of trichomonal hydrolases wasapplied to each vaginal fluid specimen. One swab from each pair ofspecimens was rubbed on a substrate film, on a Fast Garnet film, and ona 35 μL drop of threonine buffer until the buffer was absorbed into theswab (“one-phase” application of reagents). At the same time, the secondswab from each pair of specimens was treated likewise, but omitting theFast Garnet film. After ten minutes, the second swab from each pair wasrubbed on a Fast Garnet film (“two-phase” application of reagents).After another thirty seconds, the intensity of pink color that developedon both swabs was visually scored in 0.5 unit increments using the 0-9color scale presented in Table 11.1.

Results

The results are shown in Table 15.1. TABLE 15.1 Comparison ofApplication Methods Vaginal Fluid Specimens One-Phase Two-Phase Donor A1 3 Donor B 1 2 Donor C 1 3 Donor D 1 2 Donor E 1 2

The data in Table 15.1 show that all five swabs in which the diazoniumdye and substrate were applied nearly simultaneously (“one-phase”application of reagents) produced a color score of 1 after ten minutes,whereas the five swabs in which the diazonium dye was applied tenminutes after the substrate (“two-phase” application of reagents) scored2 or 3.

Interpretation

In both procedures, the intensity of the pink color produced was scoredten and a half minutes after the substrate was applied to each swab, butthe pink color was less intense when the diazonium dye was applied atthe start rather than at the end of the ten-minute period. Eitherprocedure will detect trichomonal hydrolases, but the “two-phase”procedure produces a more intense color.

Experiment 16

This experiment compared the responses of two peptide-based substratesat low pH to specimens from women attending an STD clinic, includingboth trichomonas-positive and trichomonas-negative specimens.

Materials

-   -   Dacron swabs (two per donor) containing vaginal fluid specimens        collected from women subjects, many of whom had bacterial        vaginosis, vaginal yeast infections, or other forms of        vaginitis, and which were determined to be trichomonas-positive        (vaginally infected with T vaginalis) or trichomonas-negative by        5-day culture for T. vaginalis    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation E using 200        mM ZRRR-MNA, and as described in Preparation F using 200 mM        VLR-MNA    -   Indicator films prepared as described in Preparation G using 10        mM Fast Garnet diazonium dye        Procedure

For each pair of vaginal fluid specimens from a clinic patient, one swabwas rubbed on ZRRR-MNA film and the other swab was rubbed on VLR-MNAfilm. Immediately after each swab was rubbed on a substrate film, theswab was rubbed on a 35 μL drop of threonine buffer until the buffer wasabsorbed into the swab. The swabs were left lying on the plastic for tenminutes, then each swab was rubbed on a Fast Garnet film. The intensityof pink color that developed on each swab after thirty seconds wasvisually scored in 0.5-unit increments using the 0-9 color scalepresented in Table 11.1.

Results

The results are shown in Table 16.1. TABLE 16.1 Comparison of SubstrateResponses Vaginal Fluid Specimens ZRRR-MNA VLR-MNA Trichomonas-positive(n = 8): Scoring 0 0 0 Scoring Trace 0 0 Scoring 1-2 4 4 Scoring 3-4 3 2Scoring >4 1 2 Trichomonas-negative (n = 28): Scoring 0 15 23 ScoringTrace 5 0 Scoring 1-2 6 4 Scoring 3-4 2 1 Scoring >4 0 0

The data in Table 16.1 show that both ZRRR-MNA and VLR-MNA werehydrolyzed by the hydrolases present in vaginal fluid specimens fromwomen with trichomoniasis. The intensity of pink color produced byeither peptide substrate was nearly equivalent in these pairedspecimens. For either substrate, 8 of 8 (100%) trichomonas-positiveswabs produced pink color, half scoring 1-2 and half scoring 3 orhigher. In specimens from women that did not have trichomoniasis, 13 of28 (46%) swabs treated with ZRRR-MNA produced pink color, whereas only 5of 28 (18%) swabs treated with VLR-MNA produced pink color.

Interpretation

Both peptide substrates were hydrolyzed efficiently by hydrolasespresent in vaginal fluid specimens from women with trichomoniasis, butVLR-MNA was hydrolyzed less often than ZRRR-MNA by trichomonas-negativespecimens.

Experiment 17

This experiment examines the effect of increasing the amount ofD-Val-Leu-Arg-MNA (VLR-MNA) on the intensity of pink color produced oncotton swabs containing trichomonal hydrolases.

Materials

-   -   Trichomonal hydrolases prepared as described in Preparation D    -   Pooled normal vaginal fluid supernatant prepared as follows:        vaginal fluid specimens collected on Dacron swabs from normal,        uninfected women were centrifuged to extract the undiluted fluid        from the swabs and to pellet the particulate matter, and then        the supernatants were pooled and frozen until use    -   100 mM L-cysteine hydrochloride in water    -   200 mM L-threonine buffer, pH 2.4    -   Substrate film dots prepared as described in Preparation F using        100 mM VLR-MNA    -   Indicator films prepared as described in Preparation G using 10        mM Fast Garnet diazonium dye        Procedure

Solution A, containing 10% v/v trichomonal hydrolases and 2 mML-cysteine, was prepared by mixing 20 μL of trichomonal hydrolases with4 μl of 100 mM L-cysteine and 176 μL of water, and Solution B,containing 20% v/v trichomonal hydrolases, 5% v/v normal vaginal fluidsupernatant and 2 mM L-cysteine, was prepared by mixing 40 μL oftrichomonal hydrolases with 10 μL of normal vaginal fluid supernatant, 4μL of 100 mM L-cysteine and 176 μL of water. Several 35 μL drops each ofSolution A, Solution B, and threonine buffer were individually pipettedonto discrete, separated areas on a sheet of Mylar. Individual cottonswabs were rubbed on a drop of either Solution A or Solution B, then onone, two or three dots of substrate, and then on a drop of buffer. Afterten minutes, each swab was rubbed on an indicator film. The intensity ofpink color that developed on each swab after thirty seconds was visuallyscored in 0.5 unit increments using the 0-9 color scale presented inTable 11.1.

Results

The results are shown in Table 17.1. TABLE 17.1 Comparison of SubstrateAmounts One dot of Two dots of Three dots of Test Solution SubstrateSubstrate Substrate Solution A 5.5 7 9 Solution B 2.5 4 5.5

The data in Table 17.1 show that the intensity of pink color produced bytrichomonal hydrolases increased with increasing amounts of substrate,whether or not vaginal fluid was present. Solution A, which containedtrichomonal hydrolases but no vaginal fluid, produced a moderately darkpink color scoring 5.5 when a single dot of VLR-MNA film was applied tothe swab, and a very intense dark pink color scoring 9 when a tripleamount of VLR-MNA was applied to the swab. Likewise, the intensity ofpink color produced by Solution B, which contained trichomonalhydrolases and vaginal fluid, scored 2.5 with a single dot of VLR-MNA, 4with two dots of VLR-MNA, and 5.5 with three dots of VLR-MNA.

Interpretation

The intensity of pink color produced by trichomonal hydrolases increasedwith increasing amounts of substrate, whether or not vaginal fluid waspresent. Therefore, to maximize the sensitivity of detection oftrichomonal hydrolases in vaginal fluid specimens on swabs, a highconcentration of substrate should be used. Dry films are a veryeffective means to deliver the substrate to a swab.

Experiment 18

This experiment compares two diazonium dyes in terms of colordevelopment ten minutes after substrate had been applied to swabs, usingswabs containing normal vaginal fluid specimens and swabs containingnormal vaginal fluid to which trichomonal hydrolases added. The goal wasto determine which diazonium dye maximizes the pink color produced ontrichomonas-positive swabs and minimizes the pink color produced ontrichomonas-negative swabs.

Materials

Trichomonal hydrolases prepared as described in Preparation D Cottonswabs (two per donor) containing vaginal fluid specimens collected fromnormal, uninfected women 200 mM L-threonine buffer, pH 2.4

Substrate films prepared as described in Preparation F using 200 mMVLR-MNA Indicator films prepared as described in Preparation G using 10mM Fast Garnet diazonium dye

Procedure

1. Comparison of Diazonium Dyes Using Vaginal Fluid Specimens fromNormal, Uninfected Women

Each vaginal fluid specimen was rubbed on a dried substrate film andthen rubbed on a 35-μL drop of threonine buffer until the buffer wasabsorbed into the swab. After a ten-minute wait, one swab from each pairwas rubbed on a Fast Red RL film and the other swab was rubbed on a FastGarnet film. The intensity of pink color that developed on each swabafter thirty seconds was visually scored using the 0-9 color scalepresented in Table 11.1; fractional scores such as 0.3 were used whenpink color intensities between swabs differed only slightly.

2. Comparison of Diazonium Dyes Using Trichomonas-Positive Swabs Createdby Adding Trichomonas Hydrolases to Vaginal Fluid Specimens

In this part of the experiment, 20 μL of trichomonal hydrolases wasapplied to the tip of each swab containing a vaginal fluid specimen justprior to running the assay. Otherwise, the procedure was exactly thesame as for the swabs containing only vaginal fluid.

Results

The results are shown in Table 18.1. TABLE 18.1 Comparison of DiazoniumDyes Vaginal Fluid Specimens Fast Red RL Fast Garnet Specimens fromNormal, Uninfected Women (n = 5): Scoring 0 4 5 Scoring Trace 1 0Scoring 1-2 0 0 Scoring 3-4 0 0 Scoring >4 0 0 Trichomonal hydrolasesadded (n = 5): Scoring 0 0 0 Scoring Trace 1 3 Scoring 1-2 2 1 Scoring3-4 1 1 Scoring >4 1 0

The data in Table 18.1 show that swabs containing vaginal fluid fromnormal, uninfected women produced very little pink color when treatedwith substrate and buffer, followed ten minutes later by either Fast RedRL or Fast Garnet. Four of five swabs treated with Fast Red RL, and fiveof five swabs treated with Fast Garnet, had no visible pink color whenthe swabs were examined thirty seconds after the diazonium dye wasapplied; one of the five swabs treated with Fast Red RL produced a traceamount of pink color. When a small amount of trichomonal hydrolases wasadded to vaginal fluid specimens to simulate trichomonas-positivespecimens, all of the swabs produced at least a trace amount of pinkcolor. The simulated trichomonas-positive swabs treated with Fast Red RLproduced more intense pink color than those treated with Fast Garnet. Offive simulated trichomonas-positive swabs treated with Fast Red RL, twoscored three or higher, two scored 1-2, and only one had trace pinkcolor. Of five simulated trichomonas-positive swabs treated with FastGarnet, one scored three or higher, one scored 1-2, and three had onlytrace pink color.

Interpretation

Either Fast Red RL or Fast Garnet can be used in a device to detecttrichomonal hydrolases. Both dyes produced little or no pink color inswabs that contained vaginal fluid from normal, uninfected women, andboth produced pink color in swabs that had trichomonal hydrolases addedto the vaginal fluid. However, Fast Red RL produced more intense pinkcolor than Fast Garnet in the trichomonal hydrolase-containing swabs.

Experiment 19

This is a further study of the two diazonium dyes studied in Experiment18, using however actual vaginal fluid specimens from women attending anSTD clinic.

Materials

-   -   Dacron or cotton swabs (two per donor) containing vaginal fluid        specimens collected from women attending an STD clinic, many of        whom had bacterial vaginosis, vaginal yeast infections, or other        forms of vaginitis, and which were determined to be        trichomonas-positive (vaginally infected with T. vaginalis) or        trichomonas-negative by five-day culture for T. vaginalis    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation F using 200        mM VLR-MNA    -   Indicator films prepared as described in Preparation G using        either 10 mM Fast Garnet or 70 mM Fast Red RL diazonium dye        Procedure

Each of the two vaginal fluid specimens from each clinic patient wasrubbed on a substrate film and then rubbed on a 35 μL drop of threoninebuffer until the buffer was absorbed into the swab. After a ten-minutewait, one swab from each pair was rubbed on a Fast Red RL film and theother swab was rubbed on a Fast Garnet film. The intensity of pink colorthat developed on each swab after thirty seconds was visually scored in0.5 unit increments using the 0-9 color scale presented in Table 11.1.

Results

The results are shown in Table 19.1. TABLE 19.1 Comparison of DiazoniumDyes Vaginal Fluid Specimens Fast Red RL Fast GarnetTrichomonas-positive (n = 12): Scoring 0 0 0 Scoring Trace 0 0 Scoring1-2 2 5 Scoring 3-4 6 6 Scoring >4 4 1 Trichomonas-negative (n = 39):Scoring 0 25 28 Scoring Trace 8 8 Scoring 1-2 6 3 Scoring 3-4 0 0Scoring >4 0 0

The data in Table 19.1 show that swabs containing vaginal fluidcollected from twelve women with trichomoniasis produced more intensepink color when Fast Red RL rather than Fast Garnet was used to developthe color. All of the trichomonas-positive specimens produced pink colorscores of 1 or higher regardless of which diazonium dye was used, butfour of the swabs treated with Fast Red RL scored over 4; whereas onlyone of the swabs treated with Fast Garnet scored over 4. Most of theswabs containing vaginal fluid specimens from 39 women withouttrichomoniasis produced no more than trace color with either diazoniumdye.

Interpretation

Consistently with the results Experiment 18, Fast Red RL produced moreintense pink color than Fast Garnet in trichomonas-positive swabs(containing vaginal fluid from women with trichomoniasis).

Experiment 20

This experiment compared two procedures using swabs containing vaginalfluid specimens collected from women attending an STD clinic: (a)application of the buffer after the swab is rubbed on the substratefilm; and (b) application of the buffer to the substrate film prior torubbing the swab on the film. The goal was to determine which proceduremaximizes the pink color produced on trichomonas-positive swabs andminimizes the pink color produced on trichomonas-negative swabs.

Materials

-   -   Cotton swabs (two per donor) containing vaginal fluid specimens        collected from women attending an STD clinic, many of whom had        bacterial vaginosis, vaginal yeast infections, or other forms of        vaginitis, and which were determined to be trichomonas-positive        (vaginally infected with T. vaginalis) or trichomonas-negative        by five-day culture for T. vaginalis    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation F using 200        mM VLR-MNA    -   Indicator films prepared as described in Preparation G using        either 10 mM Fast Garnet or 70 mM Fast Red diazonium dye        Procedure

One of the two vaginal fluid specimens from each clinic patient wasrubbed on a substrate film and then rubbed on a 35 μL drop of threoninebuffer until the buffer was absorbed into the swab(buffer-after-substrate procedure). The second swab from each pair wasrubbed on the substrate film immediately after a 35 μL drop of threoninebuffer was applied to the substrate film, so both the buffer and thesubstrate were simultaneously mixed into the swab (buffer-with-substrateprocedure). After a ten-minute wait, each swab was rubbed on a Fast RedRL film. The intensity of pink color: that developed on each swab afterthirty seconds was visually scored in 0.5 unit increments using the 0-9color scale presented in Table 11.1.

Results

The results are shown in Table 20.1. TABLE 20.1 Comparison ofApplication Procedures Buffer After Buffer With Vaginal Fluid Specimenssubstrate Substrate Trichomonas-positive (n = 12): Scoring 0 3 4 ScoringTrace 1 1 Scoring 1-2 3 1 Scoring 3-4 1 2 Scoring >4 4 4Trichomonas-negative (n = 31): Scoring 0 25 31 Scoring Trace 4 0 Scoring1-2 2 0 Scoring 3-4 0 0 Scoring >4 0 0

The data in Table 20.1 show that nine of the twelve trichomonas-positivespecimens processed using the buffer-after-substrate procedure producedsome pink color, with five scoring 3 or higher. Eight of the twelvetrichomonas-positive specimens processed using the buffer-with-substrateprocedure produced some pink color, with six scoring 3 or higher. Theeffect of procedure was greater for the trichomonas-negative specimens.Of the trichomonas-negative specimens, all 31 scored zero (the desirednegative test result) with the buffer-with-substrate procedure, but only25 scored zero with the buffer-after-substrate procedure.

Interpretation

Compared to the buffer-after-substrate procedure, thebuffer-with-substrate procedure diminished the number of false-positivetest results from trichomonas-negative specimens while maintaining thedesired production of pink color by trichomonas-positive specimens.

Experiment 21

Using swabs containing vaginal fluid specimens collected from womenattending an STD clinic, this experiment examined the effect ofdecreasing the test duration from ten minutes to five minutes.

Materials

-   -   Cotton swabs (two per donor) containing vaginal fluid specimens        collected from women attending an STD clinic, many of whom had        bacterial vaginosis, vaginal yeast infections, or other forms of        vaginitis, and which were determined to be trichomonas-positive        (vaginally infected with T vaginalis) or trichomonas-negative by        five-day culture for T. vaginalis    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation F using 200        mM VLR-MNA    -   Indicator films prepared as described in Preparation G using 10        mM Fast Garnet diazonium dye        Procedure

Each of the two vaginal fluid specimens from each clinic patient wasrubbed on a substrate film just after a 35 μL drop of threonine bufferhad been applied to the substrate film. After a five-minute wait, oneswab was rubbed on a Fast Red RL film. After another five minutes, thesecond swab was rubbed on a Fast Red RL film. The intensity of pinkcolor that developed on each swab after thirty seconds was visuallyscored in 0.5 unit increments using the 0-9 color scale presented inTable 11.1.

Results

The results are shown in Table 21.1. TABLE 21.1 Comparison ofApplication Procedures Vaginal Fluid Specimens 5 Minutes 10 MinutesTrichomonas-positive (n = 17): Scoring 0 2 2 Scoring Trace 1 1 Scoring1-2 7 2 Scoring 3-4 1 5 Scoring >4 6 7 Trichomonas-negative (n = 53):Scoring 0 49 43 Scoring Trace 4 8 Scoring 1-2 0 1 Scoring 3-4 0 0Scoring >4 0 0

The data in Table 21.1 show that the five-minute and ten-minute testseach produced fifteen positive test results (swabs scoring of one orhigher) from seventeen paired specimens containing vaginal fluidcollected from women with trichomoniasis. However, the intensity of pinkcolor was less intense on the swabs given the five-minute test. Twelveof seventeen trichomonas-positive swabs given ten-minute tests scored 3or higher, whereas only seven of seventeen trichomonas-positive swabsgiven five-minute tests scored 3 or higher. The five-minute testproduced fewer false-positive test results than the ten-minute test,however. Of 53 paired trichomonas-negative specimens tested, thefive-minute test produced 49 negative results (yellow swab tip) and fourtrace results, whereas the ten-minute test produced 43 negative results,eight trace results, and one weak positive result.

Interpretation

This experiment demonstrated that an accurate five-minute swab-based“rub-and-read” test for vaginal trichomoniasis can be made using a pH2.4 buffer and two dry rub-off films.

Experiment 22

This experiment compared cotton swabs with Dacron swabs in terms of theintensity of pink color, using swabs containing vaginal fluid specimenscollected from women attending an STD clinic.

Materials

-   -   Swabs (on cotton and one Dacron from each donor) containing        vaginal fluid specimens collected from women attending an STD        clinic, many of whom had bacterial vaginosis, vaginal yeast        infections, or other forms of vaginitis, and which were        determined to be trichomonas-positive (vaginally infected        with T. vaginalis) or trichomonas-negative by five-day culture        for T. vaginalis    -   200 mM L-threonine buffer, pH 2.4    -   Substrate films prepared as described in Preparation F using 200        mM VLR-MNA    -   Indicator films prepared as described in Preparation G using 10        mM Fast Garnet diazonium dye        Procedure

Each of the two vaginal fluid specimens from each clinic patient, onecollected on a cotton swab and the other on a Dacron swab, was rubbed ona substrate film just after a 35-μL drop of threonine buffer had beenapplied to the substrate film. After a five-minute wait, one swab wasrubbed on a Fast Red RL film. The intensity of pink color that developedon each swab after thirty seconds was visually scored in 0.5 unitincrements using the 0-9 color scale presented in Table 11.1.

Results

The results are shown in Table 22.1. TABLE 22.1 Comparison ofApplication Procedures Vaginal Fluid Specimens Dacron swabs Cotton swabsTrichomonas-positive (n = 22): Scoring 0 3 5 Scoring Trace 1 1 Scoring1-2 6 6 Scoring 3-4 4 2 Scoring >4 8 8 Trichomonas-negative (n = 49):Scoring 0 41 45 Scoring Trace 7 4 Scoring 1-2 1 0 Scoring 3-4 0 0Scoring >4 0 0

The data in Table 22.1 show that the pink color produced on swabscontaining vaginal fluid collected from women with trichomoniasis wassomewhat more intense when the specimens were collected on Dacron swabsrather than cotton swabs. Twelve of 22 trichomonas-positive specimenscollected on Dacron swabs scored three or higher, whereas ten of 22trichomonas-positive specimens on cotton swabs scored three or higher.False-positive test results (scores of one or higher fromtrichomonas-negative specimens) were rare with either type of swab, butcotton swabs were less likely than Dacron swabs to produce even traceamounts of pink color from trichomonas-negative specimens. Of 49trichomonas-negative specimens collected on cotton swabs, 45 produced nopink color at all (the swab tip was yellow), and four produced a traceamount of pink color (the swab tip was peach or orange). Of 49trichomonas-negative specimens collected on Dacron swabs, 41 produced nopink color at all, seven produced a trace amount of pink color, and oneproduced a weak positive result (faint pink, scoring 1).

Interpretation

Either Dacron or cotton swabs can be used to collect vaginal fluidspecimens for this diagnostic test for trichomoniasis. The test is notlimited to a specific type of swab for specimen collection.

1. A test device for assaying a sample of vaginal fluid for the presenceof a hydrolytic enzyme associated with Trichomoniasis vaginalis, saidtest device comprising: a flow passage having a defined application sitefor application of said sample; a solid porous material, retained withinsaid flow passage, that retains particulate matter greater than 20microns in diameter while allowing aqueous liquids to pass; an indicatordeposited in solid form in said flow passage, said indicator beingsoluble in an aqueous liquid and producing a detectable change whencontacted with an activated reagent; and a conjugate deposited in solidform in said flow passage, said conjugate comprising said reagentcovalently bonded to a substrate for said hydrolytic enzyme, saidreagent releasable from said substrate by enzymatic hydrolytic cleavageaction and thereby activated; at least one of said indicator and saidconjugate being spatially separated from said application site by adistance sufficiently great that a detectable change in said indicatoris caused only by hydrolytic activity of said hydrolase in a fraction ofsaid sample remaining after said particulate matter has been removedfrom said sample by said solid porous material.
 2. A test device inaccordance with claim 1 in which said application site and said solidporous material define a flow direction of said sample within saiddevice, said indicator is deposited in a first location along said flowdirection and said conjugate is deposited in a second location alongsaid flow direction downstream of said first location.
 3. A test devicein accordance with claim 1 in which said solid porous material retainsparticulate matter greater than 10 microns in diameter.
 4. A test devicein accordance with claim 1 in which said solid porous material retainsparticulate matter greater than 1 micron in diameter.
 5. A test devicein accordance with claim 1 further comprising a hydrolase inhibitordeposited in solid form in said flow passage, said hydrolase inhibitorinhibiting hydrolases other than hydrolases characteristic ofTrichomoniasis vaginalis.
 6. A test device in accordance with claim 2further comprising a hydrolase inhibitor deposited in solid form inadmixture with said indicator in said first location.
 7. A test devicein accordance with claim 5 in which said hydrolase inhibitor is a memberselected from the group consisting of antipain, chymostatin, andtrans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, the polypeptideLys-Pro-Gln-Leu-Trp-Pro, the polypeptide Arg-Lys-Asn-Val-Tyr, and thedipeptide Lys-Pro.
 8. A test device in accordance with claim 1 in whichsaid substrate is a peptide of 1 to 6 amino acids, the C-terminus ofwhich is bonded directly to said reagent and the C-terminal amino acidis a member selected from the group consisting of lysine and arginine.9. A test device in accordance with claim 1 in which said substrate is apeptide of 2 to 3 amino acids.
 10. A test device in accordance withclaim 1 in which the N-terminus of said peptide is protected againsthydrolysis by an N-blocking group.
 11. A test device in accordance withclaim 10 in which said N-blocking group is a member selected from thegroup consisting of carbobenzoxy, benzoyl, t-butoxycarbonyl, and aD-amino acid.
 12. A test device in accordance with claim 1 in which saidreagent is a member selected from the group consisting of4-methoxy-2-naphthylamine, beta.-naphthylamine,7-amino-4-methylcoumarin, .alpha.-naphthol, .beta.-naphthol,3-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid,6-hydroxy-2-naphthalenesulfonic acid, 1-naphthol-3,6-disulfonic acid,6-bromo-2-naphthol, 6-hydroxy-2-naphthyl disulfide, and4-hydroxy-1-naphthalenesulfonic acid, and said indicator is a diazoniumdye.
 13. A test device in accordance with claim 1 in which saidconjugate is a member selected from the group consisting ofcarbobenzoxy-L-valine-L-argi-nine-4-methoxy-2-naphthylamine,carbobenzoxy-L-arginine-L-arginine-4-metho-xy-2-naphthylamine,carbobenzoxy-L-arginine-L-arginine-L-arginine-4-methox-y-2-naphthylamine,carbobenzoxy-L-leucine-L-arginine-4-methoxy-2-naphthyla-mine,carbobenzoxy-L-valine-L-arginine-4-methoxy-2-naphthylamine, andD-valine-L-leucine-L-arginine-4-methoxy-2-naphthylamine, and saidindicator is a member selected from the group consisting ofp-dimethylaminocinnamaldehyde, diazonium dyes and tetrazonium dyes. 14.A test device in accordance with claim 1 in which said conjugate is amember selected from the group consisting ofcarbobenzoxy-L-arginine-L-ar-ginine-L-arginine-4-methoxy-2-naphthylamineand D-valine-L-leucine-L-argin-ine-4-methoxy-2-naphthylamine, and saidindicator is p-dimethylaminocinnamaldehyde.
 15. A test device inaccordance with claim 2 further comprising a developing reagentdeposited in solid form in said flow passage in a third location spacedapart from said first and second locations, and means for manuallyplacing said third location in contact with said second location.
 16. Atest device in accordance with claim 15 in which said indicator isp-dimethylaminocinnamaldehyde and said developing reagent is maleicacid.